Polypeptides comprising a modified bacteriophage g3p amino acid sequence lacking a glycosylation signal

ABSTRACT

The invention relates to polypeptides that comprise a portion of filamentous bacteriophage gene 3 protein (g3p) sufficient to bind to and/or disaggregate amyloid, e.g., the N1-N2 portion of g3p and mutants and fragments thereof, wherein that g3p amino acid sequence has been modified through amino acid deletion, insertion or substitution to remove a putative glycosylation signal. The invention further relates to such polypeptides that are also modified through additional amino acid substitution to be substantially less immunogenic than the corresponding wild-type g3p amino acid sequence when used in vivo. The polypeptides of the invention retain their ability to bind and/or disaggregate amyloid. The invention further relates to the use of these g3p-modified polypeptides in the treatment and/or prevention of diseases associated with misfolding or aggregation of amyloid.

This application claims the benefit of U.S. Provisional Application No.62/087,052, filed Dec. 3, 2014, which is incorporated here by referencein its entirety to provide continuity of disclosure.

The invention relates to polypeptides that comprise a portion offilamentous bacteriophage gene 3 protein (g3p) sufficient to bind toand/or disaggregate amyloid, i.e., the N1-N2 portion of g3p and mutantsand fragments thereof, wherein that g3p amino acid sequence has beenmodified through amino acid deletion, insertion or substitution toremove a putative glycosylation signal. The invention further relates tosuch polypeptides that are also modified through additional amino acidsubstitution to be substantially less immunogenic than the correspondingwild-type g3p amino acid sequence when used in vivo. The polypeptides ofthe invention retain their ability to bind and/or disaggregate amyloid.The invention further relates to the use of these g3p-modifiedpolypeptides in the treatment and/or prevention of diseases associatedwith misfolding or aggregation of amyloid.

Filamentous bacteriophage g3p protein, and in particular the polypeptideportion thereof comprising the N1-N2 region of g3p, has beendemonstrated to bind to and disaggregate various amyloids, such asß-amyloid, tau protein, and prion proteins. See co-pending PCTapplication PCT/US2012/066793, and US provisional applications U.S.61/801,349, and U.S. 61/801,849, the disclosure of each of which isincorporated herein by reference. See also, R. Krishnan et al., J. Mol.Biol. (2014). Despite that efficacy, it is expected that production ofsuch polypeptides in recombinant mammalian cell systems could bedeleteriously affected by glycosylation at a putative asparagine-linkedglycosylation signal in the g3p sequence. In addition, systemicadministration of polypeptides comprising g3p or the N1-N2 regionthereof to humans could cause a deleterious immune response. None ofthese prior art teachings identify any potential problems relating toputative glycosylation.

The efficacy of many recombinant or otherwise non-native therapeuticproteins or polypeptides may be limited by unwanted immune reactions ofpatients to the therapeutic protein or polypeptide. A principal factorin the induction of an immune response by a protein is the presence ofT-cell epitopes within the protein, i.e., amino acid sequences that canstimulate the activity of T-cells via presentation on majorhistocompatibility complex (MHC) class I molecules. T-cell epitopes arecommonly defined as any amino acid sequences with the ability to bind toMHC class II molecules. When bound to MHC molecules, T-cell epitopes canbe recognized by a T-cell receptor (TCR), and can cause the activationof T-cells by engaging a T-cell receptor to promote a T-cell response.It is, however, generally understood that certain T-cell epitopes whichbind to MHC class II molecules do not stimulate T-cell response, becausethese peptides are recognized as “self” within the organism to which theprotein is administered.

Some T-cell epitopes may be released as peptides during the degradationof the therapeutic protein or polypeptide within cells and thenpresented by molecules of the MHC to trigger the activation of T-cells.For peptides presented by MHC class II molecules, such activation ofT-cells can then give rise, for example, to an antibody response bydirect stimulation of B-cells to produce such antibodies.

MHC class I molecules are a group of highly polymorphic proteins whichplay a central role in helper T-cell selection and activation. The humanleukocyte antigen group DR (HLA-DR) are the predominant isotype of thisgroup of proteins. However, isotypes HLA-DQ and HLA-DP perform similarfunctions. In humans approximately 70 different allotypes of the DRisotype are known, for DQ there are 30 different allotypes and for DP 47different allotypes are known. Each individual bears two to four DRalleles, two DQ and two DP alleles.

The immune response to a protein or polypeptide in an individual isheavily influenced by T-cell epitope recognition which is a function ofthe peptide binding specificity of that individual's HLA-DR allotype. Inorder to identify T-cell epitopes within a protein or polypeptide in thecontext of a global population, it is desirable to consider the bindingproperties of as diverse a set of HLA-DR allotypes as possible, thuscovering as high a percentage of the world population as possible.

T-cell epitope identification is the first step to epitope elimination.Methods enabling the detection of T-cell epitopes are known in the artand are disclosed in WO 98/52976, WO 00/34317, US2007/0269435; U.S. Pat.No. 7,208,147, Kern et al., Nature Medicine 4:975-978 (1998); and Kwoket al., Trends in Immunology 22:583-588 (2001). In these approaches,predicted or identified T-cell epitopes are removed by the use ofjudicious amino acid substitutions within the primary sequence of thetherapeutic protein or polypeptide. Although these references enableputative identification of T-cell epitopes, the selection of amino acidsubstitutions that avoid negative impact on biological activity cannotbe reasonably predicted. That can only be determined by testing each ofthe modified polypeptides for such activity.

Thus, it would be desirable to examine and reduce the glycosylation,either alone or together with reducing the immunogenicity of the N1-N2portion of g3p without destroying its amyloid-binding/disaggregationproperties. This would allow the polypeptide comprising the N1-N2portion of g3p to be made in mammalian cells without the manufacturingdifficulties associated with glycosylation. In addition, reducedimmunogenicity will allow a polypeptide comprising that N1-N2 portion tobe chronically administered systemically for therapeutic and/ordiagnostic purposes. The present invention meets this need, byidentifying the putative glycosylation signal in the N1-N2 portion ofg3p and providing modifications thereof that prevent glycosylation whilepreserving activity of g3p polypeptides, as described in PCT/US13/62862(WO 2014/055515) or of g3p polypeptides that have been modified toreduce or eliminate immunogenicity as described in PCT/US2014/039760,both of which are incorporated herein by reference. Thus, certain g3ppolypeptides of the invention are not only modified to preventglycosylation, but also comprise specific amino acids substitutionswithin these potential T-cell epitopes to produce a variant N1-N2sequence that will reduce or eliminate the immunogenicity of that T-cellepitope without destroying the ability of the variant N1-N2 to bind toamyloid, prevent amyloid aggregation, and/or effect disaggregation ofamyloid plaques.

In certain embodiments of the invention, the polypeptides comprise g3por an amyloid binding fragment thereof that has been modified to removea glycosylation signal. In one embodiment, the invention also providespolypeptides comprising a variant of an N1-N2 amino acid sequence, or amutant or fragment thereof, having reduced immunogenicity due to one ormore amino acid substitutions within one or more of the identifiedT-cell epitopes and lacking a glycosylation signal. In one aspect, theinvention provides fusion proteins comprising the variant N1-N2 sequencefused to a human immunoglobulin Fc region.

In another embodiment, the invention provides pharmaceuticalcompositions comprising the polypeptides of the invention and methods oftreating or preventing diseases associated with misfolded and/oraggregated amyloid proteins by administering such pharmaceuticalcompositions to a subject suffering from or susceptible to such disease.

Ina further embodiment, the invention provides nucleic acid moleculeswhich code for the polypeptides of the invention, as well as vectorscomprising those nucleic acid molecules and cells harboring suchvectors.

In another embodiment, the invention provides methods for producing thepolypeptides of the invention. In particular, such methods employ thenucleic acid molecules and/or cells harboring a vector that comprisessuch nucleic acid molecules.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 presents the amino acid sequence of an N1-N2-hIgG1-Fc fusionprotein (SEQ ID NO:1) with five T-cell epitopes identified by bold andunderline and the putative glycosylation signal italicized, bolded andunderlined. Amino acids 1-217 constitute the N1-N2 portion of thewild-type g3p sequence. Amino acids 218-256 represent a linker regionconsisting of the wild-type g3p glycine-rich N2-C-terminal linkerpresent in M13 bacteriophage. This region is identified by shading.Amino acids 257-261 represent amino acids encoded by the multiplecloning site used to construct a nucleic acid molecule encoding thefusion protein. The IgG-Fc portion of the protein begins at amino acid262.

FIG. 2 presents the amino acid sequence of another g3p-hIgG1-Fc fusionprotein (SEQ ID NO:2) with three T-cell epitopes identified by bold andunderline and the putative glycosylation signal italicized, bolded andunderlined. The fourth T-cell epitope has been eliminated bysubstitution of V215A and G220E as compared to SEQ ID NO:1 and the fifthT-cell epitope has been eliminated by deletion of amino acidscorresponding to amino acids 258 and 259 of SEQ ID NO:1.

FIG. 3 presents a DNA sequence (SEQ ID NO:3) encoding the g3p-hIgG1-Fcfusion protein of SEQ ID NO:1 with a N-terminal mammalian signalsequence.

FIG. 4 presents a DNA sequence (SEQ ID NO:4) encoding the g3p-hIgG1-Fcfusion protein of SEQ ID NO:2 with a N-terminal mammalian signalsequence.

FIG. 5 provides a comparison of the frequency of donor allotypesexpressed in the study described in Example 1.

FIG. 6 presents the amino acid sequence (SEQ ID NO:5) of a polypeptideof the invention that is a g3p-hIgG1-Fc fusion protein with amino acidchanges in the putative glycosylation signal (T41G), epitope 1 (T56H),and epitope 3 (K174R), as compared to SEQ ID NO:2. The substituted aminoacids are bolded, italicized, underlined and by gray highlighting.

FIG. 7 presents a DNA sequence (SEQ ID NO:6) of a plasmid encodingPolypeptide 86 with a N-terminal mammalian signal sequence. The codingsequence, including the signal sequence is indicated by bolding. Thecodon changes as compared to the DNA encoding SEQ ID NO:1 are indicatedby underlining.

FIG. 8 presents a DNA sequence (SEQ ID NO:7) of a plasmid encodingPolypeptide 86 T41G with a N-terminal mammalian signal sequence. Thecoding sequence, including the signal sequence is indicated by holding.The codon changes as compared to the DNA encoding SEQ ID NO:1 areindicated by underlining.

FIG. 9 depicts a SDS-PAGE analysis comparing the polypeptide of SEQ IDNO:1 with Polypeptide 86 and Polypeptide 86 T41G.

FIG. 10 depicts the results a filter retardation assay comparing thepolypeptide of SEQ ID NO:1 with Polypeptide 86 and Polypeptide 86 T41G.

FIG. 11 depicts comparative binding of a polypeptide of SEQ ID NO:1 withPolypeptide 86 T41G for three different types of fibers as measured byELISA.

DETAILED DESCRIPTION OF THE INVENTION

In the present application the term “variant” (and its cognates) withrespect to a reference (unmodified) amino acid or nucleic acid sequencerefers to a sequence that contains one or more amino acidssubstitutions, deletions or insertions, or corresponding substitution,deletion or insertion of codons. The reference sequence is also referredto as a “starting amino acid sequence” or “starting sequence.” A variantdoes not necessarily require physical manipulation of the referencesequence. As long as a sequence contains a different amino acid ascompared to a reference sequence it will be considered a “variant”regardless of how it was synthesized.

As used herein, the term “mutant” (and its cognates) refers to astarting sequence that has been modified as compared to a specificsequence set forth in the application (e.g., SEQ ID NO:1, SEQ ID NO:2,etc).

As used herein, the term “modified” (and its cognates) refers to changein a reference amino acid sequence or nucleic acid sequence. When astarting sequence is modified the resulting sequence is a variant. Amodification includes one or more amino acids substitutions, deletionsor insertions, or corresponding substitution, deletion or insertion ofcodons.

The term “corresponding substitution” as used herein means asubstitution in a mutant or fragment of amino acids 1-217 of SEQ ID NO:1that corresponds to the equivalent amino acid substitution in Table 1,Table 2, Table 6 or Table 7 when such mutant or fragment is aligned withamino acids 1-217 of SEQ ID NO:1.

An example of a fragment of amino acids 1-217 of SEQ ID NO:1 that bindsto and/or disaggregates amyloid includes, but is not limited to, anyfragment that comprises amino acids 1-67 of SEQ ID NO:1. Example ofmutants of amino acids 1-217 of SEQ ID NO:1 that bind to and/ordisaggregate amyloid include, but are not limited to: (1) amino acids1-217 of SEQ ID NO:2; (2) amino acids 1-217 of SEQ ID NO:1, amino acids1-217 of SEQ ID NO:2, or amino acids 1-217 of SEQ ID NO:5 bearingsubstitution of VVV at amino acids 43-45 with AAA; (3) amino acids 1-217of SEQ ID NO:1, amino acids 1-217 of SEQ ID NO:2, or amino acids 1-217of SEQ ID NO:5 having the substitution C53W; (4) amino acids 1-217 ofSEQ ID NO:1, amino acids 1-217 of SEQ ID NO:2, or amino acids 1-217 ofSEQ ID NO:5 having a deletion of amino acids 96-103; (5) amino acids1-217 of SEQ ID NO:1, amino acids 1-217 of SEQ ID NO:2, or amino acids1-217 of SEQ ID NO:5 bearing the substitution of QPP at amino acids212-214 with AGA; (6) amino acids 1-217 of SEQ ID NO:1, amino acids1-217 of SEQ ID NO:2, or amino acids 1-217 of SEQ ID NO:5 having thesubstitutions W181A, F190A and F194A; (7) other active mutants andfragments disclosed in PCT/US2012/066793; (8) amino acids 1-217 of SEQID NO:5; (9) amino acids 2-217 of SEQ ID NO:1, amino acids 2-217 of SEQID NO:2, or amino acids 2-217 of SEQ ID NO:5; (10) amino acids 3-217 ofSEQ ID NO:1, amino acids 3-217 of SEQ ID NO:2, or amino acids 3-217 ofSEQ ID NO:5; (11) any one of amino acids 1-217 of SEQ ID NO:1, aminoacids 1-217 of SEQ ID NO:2, or amino acids 1-217 of SEQ ID NO:5containing an additional N-terminal methionine residue.

The N1-N2 portion of filamentous bacteriophage g3p protein haspreviously been shown to possess amyloid binding and disaggregationproperties (see PCT/US2012/066793). The N1-N2 portion of native M13phage is represented by amino acids 1-217 of SEQ ID NO:1. The same N1-N2amino acid sequence is also present in fd and fl filamentousbacteriophage. It should be understood that amino acids 218-256 of SEQID NO:1 are also part of the native g3p sequence and are typicallyreferred to as the glycine-rich linker connecting the N2 region of g3pto the C-terminal domain of g3p (CT), also known as the N3 domain. Aminoacids 257-261 of SEQ ID NO:1 represent amino acids encoded by themultiple cloning site used to construct a nucleic acid molecule encodingthe fusion protein of SEQ ID NO:1.

Polypeptides

Thus, in one embodiment, the invention provides a polypeptide comprisinga variant of a starting amino acid sequence, wherein the starting aminoacid sequence is selected from: amino acids 1-217 of SEQ ID NO:1 oramino acids 1-217 of SEQ ID NO:2 and mutants of any of the foregoinghaving one or more of the following modifications: substitution of VVVat amino acids 43-45 with AAA; substitution C53W; deletion of aminoacids 96-103; substitution of QPP at amino acids 212-214 with AGA;substitutions W181A, F190A and F194A; deletion of amino acid 1; deletionof amino acids 1 and 2; and addition of a N-terminal methionine residue,wherein:

-   -   (a) the starting amino acid sequence is modified to remove the        putative glycosylation signal at amino acids 39-41; and    -   (b) the polypeptide binds to and/or disaggregates amyloid.

In one aspect of this embodiment, the starting amino acid sequence isselected from: amino acids 1-217 of SEQ ID NO:1, and amino acids 1-217of SEQ ID NO:2.

Elimination of the putative glycosylation signal is achieved by aminoacid substitution of one or more of N39, A40 and/or T41; deletion of oneor more of N39, A40 and/or T41; insertion of one or more amino acidsbetween N39 and A40; and insertion of one or more amino acids betweenA40 and T41 insofar as such substitution, deletion or insertion does notregenerate a glycosylation signal. The putative glycosylation sequenceis Asn-X-Thr/Ser, wherein X is any amino acid other than Pro or Cys.Thus, only certain substitutions for A40 will eliminate theglycosylation sequence. In one aspect of these embodiments eliminationof the putative glycosylation signal is achieved by amino acidsubstitution of one or more of N39, A40 and/or T41. In a more specificaspect of these embodiments elimination of the putative glycosylationsignal is achieved by amino acid substitution of T41. In an even morespecific aspect of these embodiments elimination of the putativeglycosylation signal is achieved by any of the following substitutions:T41G, T41W, T41H, T41V, T41I, T41L, T41R, T41K, T41Y, T41F, T41D, T41E,T41Q, T41N, or T41A. In the most specific aspect of these embodiments,elimination of the putative glycosylation signal is achieved by a T41substitution.

In another embodiment of the invention the polypeptide that has beenmodified to remove the putative glycosylation signal at amino acids39-41 additionally has reduced immunogenicity as compared to acorresponding polypeptide comprising the starting amino acid sequence;and the variant has from 1 to 9 amino acid substitutions (in addition toany substitutions that have eliminated the glycosylation signal) ascompared to the starting amino acid sequence, wherein each amino acidsubstitution is selected from the group of amino acid substitutions setforth in Table 1 and Table 2. The term “corresponding polypeptidecomprising the starting amino acid sequence” as used herein means a

polypeptide which, except for the modification of the glycosylationsignal and the additional substitution(s), has the same amino acidsequence as the polypeptide comprising the starting amino acid sequence.

TABLE 1 Deimmunizing Amino Acid Substitutions to Amino Acids 1-217 ofSEQ ID NO: 1 or SEQ ID NO: 3. Amino Acid present at Amino the indicatedAmino Epitope Acid # Acid # of SEQ ID NO: 1* Substitution 1 48 G H, K,R, S, T 1 51 T G, H. K, R, P, Q, N 1 54 Y G, H, K, R, P 1 56 T G, H, K,R, P 2 135 M A, D, G, K, N, T, H, R 2 140 R D, E, H, Q, A, G 2 141 F D,E 2 143 N A, G 3 173 S G, P, K 3 174 K R 3 176 M G, H, K, N, R 3 178 DG, N, Q, S, T 3 181 W G, H, K, R

TABLE 2 Alternate or Additional De-Immunizing Amino Acid Substitutionsto Amino Acids 1-217 of SEQ ID NO: 1, or SEQ ID NO: 3. Amino Acidpresent at Amino the indicated Amino Epitope Acid # Acid # of SEQ ID NO:1* Substitution 1 48 G D, P 1 50 E G, H, K, P, R 1 51 T W 1 53 C F, H,K, N, Q, R, W, Y 2 135 M C, E, P, Q, S 2 137 Q D, E 2 138 N D, E, G, H,P, Q, S, T 2 140 R M, N, P, S, Y 2 141 F G, N, P, Q, Y 3 173 S D, H, R,T 3 175 A G, H, K, P, R 3 176 M P, Q, W 3 178 D F, H, K, R, W, Y 3 179 AH, K, P, R 3 181 W P*In Tables 1 and 2, each of the indicated amino acids is the same in SEQID NOS: 1 and 3.

The amino acid substitutions set forth in Tables 1 and 2 were derived byidentifying the T-cell epitopes present completely within the N1-N2amino acid sequence. This was done by incubating different overlappingpeptide portions of the N1-N2 sequence against the peripheral bloodmononuclear cells (PBMC) from a cohort of community blood donors bestrepresenting the world population of HLA-DR allotypes to identify thepotential T-cell epitopes. This information was then subjected tosoftware analysis against a database of known T-cell epitopes toidentify optimal amino acid substitutions within those potentialepitopes. These procedures are described in detail in the Examples.

In one aspect of these embodiments, the 1-9 additional amino acidsubstitutions (in addition to any substitutions that have eliminated theglycosylation signal) are selected from those set forth in Table 1. In amore specific aspect of the embodiment set forth above, the polypeptidecomprises a variant of amino acids 1-217 SEQ ID NO:1 or a variant ofamino acids 1-217 of SEQ ID NO:2 having only a specific single aminoacid substitution, wherein the substitution is selected front one of thesubstitutions set forth in Table 3:

TABLE 3 Specific De-Immunizing Single Amino Acid Substitutions in AminoAcids 1-217 of SEQ ID NO: 1, or Amino Acids 1-217 of SEQ ID NO: 2 G48HG48K G48R G48S G48T T51G T51H T51K T51P T51R T51Q T51N Y54G Y54H Y54KY54P Y54R T56G T56H T56K T56P T56R M135A M135D M135G M135H M135K M135NM135R M135T R140A R140D R140E R140G R140H R140Q F141D F141E N143A N143GS173G S173P M176G M176H M176K M176N D178G D178N D178Q D178S W181G W181HW181K W181R S173K K174R M176R D178T

In an even more specific aspect of these embodiments, the specificsingle amino acid substitution is not in epitope 2 (amino acids 135-143of SEQ ID NO:1).

In some embodiments, the polypeptide comprises a variant of amino acids1-217 SEQ ID NO:1 or a variant of amino acids 1-217 of SEQ ID NO:2having 2-9 amino acid substitutions (in addition to any substitutionsthat have eliminated the glycosylation signal), wherein thesubstitutions are in at least two of epitopes 1, 2 and 3, and whereinthe substitutions are selected from those set forth in Tables 1 and 2.In a more specific aspect, at least two substitutions in the variant ofamino acids 1-217 SEQ ID NO:1 or the variant of amino acids 1-217 of SEQID NO:2 are selected from those set forth in Table 1. In an even morespecific aspect the polypeptide comprises a variant of SEQ ID NO:1 orSEQ ID NO:2 that has only two amino acid substitutions, wherein thesubstitutions are selected from any of the specific two amino acidsubstitutions set forth in Table 4:

TABLE 4 Specific De-Immunizing Two Amino Acid Substitutions in AminoAcids 1-217 of SEQ ID NO: 1, or Amino Acids 1-217 of SEQ ID NO: 2: Y54Kand M135K Y54K and M135T Y54K and R140Q Y54R and M135K Y54R and M135TY54R and R140Q T56H and M135K T56H and Ml35T T56H and R140Q T56K andM135K T56K and M135T T56K and R140Q Y54K and D178N Y54K and W181H Y54Kand W181R Y54K and K174R Y54R and D178N Y54R and W181H Y54R and W181RY54R and K174R T56H and D178N T56H and W181H T56H and W181R T56H andK174R T56K and D178N T56K and W181H T56K and W181R T56K and K174R M135Kand D178N M135K and W181H M135K and W181R M135K and K174R M135T andD178N M135T and W181H M135T and W181R M135T and K174R R140Q and D178NR140Q and W181H R140Q and W181R R140Q and K174R

Ina more specific aspect of these embodiments, neither of the two aminoacid substitutions are in epitope 2 (amino acids 135-143 of SEQ IDNO:1). In an even more specific aspect of these embodiments, the twoamino acid substitutions are T56H and K174R.

In another embodiment, the polypeptide comprises a variant of aminoacids 1-217 SEQ ID NO:1 or a variant of amino acids 1-217 of SEQ IDNO:2, having 3-9 amino acid substitutions (in addition to anysubstitutions that have eliminated the glycosylation signal), wherein atleast one amino acid substitution is in each of epitopes 1, 2 and 3, andwherein the substitutions are selected from substitutions set forth inTable 1 and Table 2. In a more specific aspect, at least three aminoacids substitution in the variant of amino acids 1-217 SEQ ID NO:1 orthe variant of amino acids 1-217 of SEQ ID NO:2 are selected fromsubstitutions set forth in Table 2. In an even more specific aspect, thepolypeptide comprising the variant of amino acids 1-217 SEQ ID NO:1 orthe variant of amino acids 1-217 of SEQ ID NO:2 has only three aminoacid substitutions wherein the substitutions are selected from any ofthe specific three amino acid substitutions set forth in Table 5.

TABLE 5 Specific De-Immunizing Three Amino Acid Substitutions in AminoAcids 1-215 of SEQ ID NO: 1, Amino Acids 1-217 of SEQ ID NO: 2: Y54K,M135K and Y54K, M135T and Y54K, R140Q and Y54R, M135K and D178N D178ND178N D178N Y54R, M135T and Y54R, R140Q and T56H, M135K and T56H, M135Tand D178N D178N D178N D178N T56H, R140Q and T56K, M135K and T56K, M135Tand T56K, R140Q and D178N D178N D178N D178N Y54K, M135K and Y54K, M135Tand Y54K, R140Q and Y54R, M135K and W181H W181H W181H W181H Y54R, M135Tand Y54R, R140Q and T56H, M135K, and T56H, M135T and W181H W181H W181HW181H T56H, R140Q and T56K, M135K and T56K, M135T and T56K, R140Q andW181H W181H W181H W181H Y54K, M135K and Y54K, M135T and Y54K, R140Q andY54R, M135K and W181R W181R W181R W181R Y54R, M135T and Y54R, R140Q andT56H, M135K and T56H, M135T and W181R W181R W181R W181R T56H, R140Q andT56K, M135K and T56K, M135T and T56K, R140Q and W181R W181R W181R W181RY54K, M135K and Y54K, M135T and Y54K, R140Q and Y54R, M135K and K174RK174R K174R K174R Y54R, M135T and Y54R, R140Q and T56H, M135K and T56H,M135T and K174R K174R K174R K174R T56H, R140Q and T56K, M135K and T56K,M135T and T56K, R140Q and K174R K174R K174R K174R

In another embodiment, the invention provides a polypeptide comprising ag3p variant wherein one of the 1 to 9 substitution is a substitution inepitope 4 selected from V215A, V215S, V215G or V215T, V215C, V215D,V215E, V215F, V215H, V215K, V215N, V215P, V215Q, or V215R. In stillanother embodiment, the invention provides a polypeptide comprising avariant of amino acids 1-217 of SEQ ID NO:1, wherein one of the 1 to 9substitutions is a substitution in epitope 4 selected from V215A, V215S,V215G, V215T, V215C, V215D, V215E, V215F, V215H, V215K, V215N, V215P,V215Q, or V215R. Through testing of overlapping potential T-cell epitopepeptide portions of the N1-N2 sequence, applicants have determined thatV215 in SEQ ID NO:1 is part of a potential T-cell epitope (epitope 4 inFIG. 1) spanning amino acids 215-223 of SEQ ID (the end of N2 through aportion of the glycine-rich linker). In a more specific aspect of thisembodiments, epitope 4 has a V215A and G220E substitution (as in SEQ IDNO:2). In addition, a single V215G substitution in epitope 4 as comparedto SEQ ID NO:1 did not affect the ability of the polypeptide to bind toor disaggregate amyloid. Each of the other substitutions for V215 setforth above are similarly predicted by software and database analysis toeliminate the T-cell epitope, while having little or no effect onamyloid binding.

In a more specific aspect, the polypeptide comprising a variant of aminoacids 1-217 SEQ ID NO:1 has a modification that removes the putativeglycosylation site at amino acids 39-41; any one of the V215substitutions set forth above; as well as 1-8 of the amino acidsubstitutions set forth in Table 1 or Table 2. In an even more specificembodiment, the 1-8 amino acid substitutions are selected from those setforth in Table 1. In an even more specific aspect, the polypeptide has aT41 substitution selected from T41G, T41W, T41H, T41V, T41I, T41L, T41R,T41K, T41Y, T41F, T41D, T41E, T41Q, T41N, and T41A; a V215 substitutionselected from V215A, V215S, V215G or V215T, V215C, V215D, V215E, V215F,V215H, V215K, V215N, V215P, V215Q, and V215R; and one additional singleamino acid substitution selected from those set forth in Table 3,wherein the single amino acid substitution is not in epitope 2. In amore specific aspect, the polypeptide comprising a variant of aminoacids 1-217 SEQ ID NO:1 has a modification that removes the putativeglycosylation site at amino acids 39-41; any one of the V215substitutions set forth above; and 2-8 additional amino acidsubstitutions, wherein the additional substitutions are in at least twoof epitopes 1, 2 and 3, and wherein the substitutions are selected fromthose set forth in Table 1 or Table 2. In an even more specificembodiment, the at least one substitution in at least two of epitopes 1,2 and 3, is selected from the substitutions set forth in Table 1. In astill more specific embodiment, the polypeptide has a T41 substitutionselected from T41G, T41W, T41H, T41V, T41I, T41L, T41R, T41K, T41Y,T41F, T41D, T41E, T41Q, T41N, and T41A; a V215 substitution selectedfrom V215A, V215S, V215G or V215T, V215C, V215D, V215E, V215F, V215H,V215K, V215N, V215P, V215Q, and V215R; and one of the specific two aminoacid substitutions set forth in Table 4, wherein the neither of theamino acid substitutions are in epitope 2.

In a more specific aspect, the polypeptide comprising a variant of aminoacids 1-217 of SEQ ID NO:1 has a modification that removes the putativeglycosylation site at amino acids 39-41; any one the V215 substitutionsset forth above; and 3-8 additional amino acid substitutions selectedfrom those set forth in Table 1 or Table 2, wherein each of epitopes 1,2 and 3, comprise one of the additional substitutions. In an even morespecific embodiment, the substitution in each of epitopes 1, 2 and 3, isselected from those set forth in Table 1. In a still more specificembodiment, the polypeptide has a T41 substitution selected from T41G,T41W, T41H, T41V, T41I, T41L, T41R, T41K, T41Y, T41F, T41D, T41E, T41Q,T41N, and T41A; a V215 substitution selected from V215A, V215S, V215G orV215T, V215C, V215D, V215E, V215F, V215H, V215K, V215N, V215P, V215Q,and V215R; an optional G220E substitution; and one of the specific threeamino acid substitutions set forth in Table 5.

In an even more specific embodiment, the polypeptide comprises a variantof amino acids 1-217 of SEQ ID NO:2 having a T41 substitution selectedfrom T41G, T41W, T41H, T41V, T41I, T41L, T41R, T41K, T41Y, T41F, T41D,T41E, T41Q, T41N, and T41A; and a pair of substitutions selected fromthose set forth in Table 4, wherein one of the substitutions is inepitope 1 and the other is in epitope 3. In one aspect of thisembodiment, the T41 substitution is T410. In an alternate aspect of thisembodiment, the pair of substitutions, wherein one of the substitutionsis in epitope 1 and the other is in epitope 3 is T56H and K174R. In aneven more specific aspect of this embodiment, the polypeptide comprisesamino acids 1-215 of SEQ ID NO:5. In another embodiment, the polypeptideof the invention is a fusion protein consisting essentially of a humanor humanized immunoglobulin Fc polypeptide sequence fused via a peptidelinker or directly to the C-terminus of the variant g3p amino acidsequence. The term “peptide linker” as used herein refers to a series ofconsecutive amino acids that will not interfere with the function of thepolypeptide. As set forth above, in SEQ ID NOs: 1 and 3, amino acids218-256 represent the glycine-rich linker that is normally present inthe M13 g3p protein. That linker may be used or a different linker maybe substituted therefor in the polypeptides of the invention.Alternatively, the Fc polypeptide sequence may be linked directly to thelast amino acid encoding N2 (e.g., amino acid 217 of SEQ ID NOs 1 and3). The choice of linker sequence and/or its absence may be made bythose of skill in the art taking into account vectors available for therecombinant expression of the polypeptide of the invention, and anysecondary or tertiary structure such a linker may impart to thepolypeptide. In one aspect of this embodiment, the Fc polypeptide is theFc portion of a human IgG. In a more specific aspect the polypeptide isa variant of SEQ ID NO:1 or SEQ ID NO:3 having a modification thatremoves the putative glycosylation site at amino acids 39-41. In an evenmore specific aspect, the polypeptide is a variant of SEQ ID NO:1 or SEQID NO:2 having a modification that removes the putative glycosylationsite at amino acids 39-41; and 1 to 9 additional amino acid residuesubstitutions therein selected from the group of amino acidsubstitutions set forth in Table 1, Table 2, or Table 6, or Table 7,below:

TABLE 6 Deimmunizing Amino Acid Substitutions to Amino Acids 215-223 ofSEQ ID NO: 1. Amino Acid present in Amino Amino Acids 1-215 of EpitopeAcid # SEQ ID NO: 1 Substitution 4 215  V* A*, S, G, T 4 218 G C, E, N,P, Q, S, T 4 220  G* E*, D, F, W 4 221 S D, E, G 4 223 G D, P

TABLE 7 Alternate and Additional Deimmunizing Amino Acid Substitutionsto Amino Acids 215-223 of SEQ ID NO: 1. Amino Acid present in AminoAmino Acids 1-215 of Epitope Acid # SEQ ID NOs 1-3 Substitution 4 215 V* C, D, E, F, H, K, N, P, Q, R 4 218 G A, H, W 4 220  G* M, Y 4 223 GE, K, N, R, T *V215A and G220E are already substituted in SEQ ID NO: 2so that a variant of SEQ ID NO: 2 would not contain a furthersubstitution at these amino acid residues.

In one embodiment, the polypeptide is a variant of SEQ ID NO:1 having amodification that removes the putative glycosylation site at amino acids39-41; and 2-9 additional amino acid substitutions, wherein one of theadditional substitutions is a substitution set forth in Table 6 andTable 7; and at least one other of the substitutions is a substitutionset forth in Table 1 and Table 2. In a more specific aspect of thisembodiment, one of the additional substitutions is a substitution setforth in Table 6; and at least one other of the substitutions is asubstitution set forth in Table 1. In an even more specific aspect ofthis embodiment, the polypeptide does not have a substitution in epitope2. In another even more specific aspect of this embodiment, thepolypeptide has a T41 substitution selected from T41G, T41W, T41H, T41V,T41I, T41L, T41R, T41K, T41Y, T41F, T41D, T41E, T41Q, T41N, and T41A. Ina still more specific aspect of this embodiment, the polypeptide has aT41G substitution.

In another embodiment, the polypeptide is a variant of SEQ ID NO:1having a modification that removes the putative glycosylation site atamino acids 39-41; and has 3-9 additional amino acid substitutions,wherein at least one of the additional substitutions is selected fromsubstitutions set forth in Table 6 and Table 7, and wherein at least twoof epitopes 1, 2, and 3 contain at least one substitution selected fromthe substitutions set forth in Table 1 and Table 2. In a more specificaspect, the polypeptide has at least one of the substitutions set forthin Table 6 and at least one substitution in at least two of epitopes 1,2 and 3 selected from the substitutions set forth in Table 1. In an evenmore specific aspect of this embodiment, the polypeptide does not have asubstitution in epitope 2. In another even more specific aspect of thisembodiment, the polypeptide has a T41 substitution selected from T41G,T41W, T41H, T41V, T41I, T41L, T41R, T41K, T41Y, T41F, T41D, T41E, T41Q,T41N, and T41A. In a still more specific aspect of this embodiment, thepolypeptide has a T41G substitution. In an alternate aspect of thisembodiment, the polypeptide has only two additional substitutions,wherein on is in epitope 1 and the other is in epitope 3. In an evenmore specific aspect of this embodiment, the polypeptide has only twoadditional substitutions, wherein one is T56H and the other is K174R.

In another embodiment, the polypeptide is a variant of SEQ ID NO:1having a modification that removes the putative glycosylation site atamino acids 39-41; and has 4-9 amino acid substitutions; at least one ofsubstitutions set forth in Table 6 and Table 7; and at least onesubstitution in each of epitopes 1, 2 and 3 selected from thesubstitutions set forth in Table 1 and Table 2. In a specific aspect ofthis embodiment, the polypeptide has at least one of the substitutionsset forth in Table 6 and at least one substitution in each of epitopes1, 2 and 3 selected from those set forth in Table 1. In another morespecific aspect, the polypeptide has at least one of substitutions setforth in Table 6; and at least one of the specific substitutions one,two or three amino acid substitutions set forth in Table 3, Table 4 orTable 5, respectively. In still another more specific aspect of thisembodiment, the polypeptide is a variant of SEQ ID NO:1 and has only oneof the amino acid substitutions set forth in Table 6 and only one, twoor three additional amino acid substitutions selected from one of thespecific one, two or three amino acid substitutions set forth in Table3, Table 4 or Table 5, respectively. In another even more specificaspect of this embodiment, the polypeptide has a T41 substitutionselected from T41G, T41W. T41H, T41V, T41I, T41L, T41R, T41K, T41Y,T41F, T41D, T41E, T41Q, T41N, and T41A. In a still more specific aspectof this embodiment, the polypeptide has a T41G substitution.

In an alternate embodiment, the polypeptide is a variant of SEQ ID NO:2having a modification that removes the putative glycosylation site atamino acids 39-41; and 1 to 9 additional amino acid residuesubstitutions selected from the group of amino acid substitutions setforth in Table 1, and Table 2. In a more specific aspect, at least oneadditional substitution is set forth in Table 1. In an even morespecific aspect of this embodiment, the polypeptide does not have asubstitution in epitope 2. In another even more specific aspect of thisembodiment, the polypeptide has a T41 substitution selected from T41G,T41W, T41H, T41V, T41I, T41L, T41R, T41K, T41Y, T41F, T41D, T41E, T41Q,T41N, and T41A. In a still more specific aspect of this embodiment, thepolypeptide has a T41G substitution.

In another embodiment, the polypeptide is a variant of SEQ ID NO:2having a modification that removes the putative glycosylation site atamino acids 39-41; and 2-9 additional amino acid substitutions and atleast one substitution in at least two of epitopes 1, 2 and 3 selectedfrom any of the substitutions set forth in Table 1 and 2. In a morespecific aspect, at least one substitution in at least two of epitopes1, 2 and 3 is selected from those set forth in Table 1. In an even morespecific aspect of this embodiment, the polypeptide does not have asubstitution in epitope 2. In another even more specific aspect of thisembodiment, the polypeptide has a T41 substitution selected from T41G,T41W, T41H, T41V, T41I, T41L, T41R T41K, T41Y, T41F, T41D, T41E, T41Q,T41N, and T41A. In a still more specific aspect of this embodiment, thepolypeptide has a T41G substitution. In an alternate aspect of thisembodiment, the polypeptide comprises at least one amino acidsubstitution in epitope 1 and at least one amino acid substitution inepitope 3. In an even more specific aspect of this embodiment, thepolypeptide comprises a T56H and a K174R substitution.

In another more specific aspect, the polypeptide is a variant of SEQ IDNO:2 or SEQ ID NO:5 and has 3-9 amino acid substitutions, wherein atleast one substitution is in each of epitopes 1, 2 and 3 and is selectedfrom any of the substitutions set forth in Table 1 and 2. In a morespecific aspect, the at least one substitution in each of epitopes 1, 2and 3 is selected from those set forth in Table 1. In an even morespecific embodiment, the polypeptide is a variant of SEQ ID NO:2 or SEQID NO:5 and has only one, two or three amino acid substitutions selectedfrom one of the specific one, two or three amino acid substitutions setforth in Table 3, Table 4 or Table 5, respectively. In another even morespecific aspect of this embodiment, the polypeptide has a T41substitution selected from T41G, T41W, T41H, T41V, T41T, T41L, T41R,T41K, T41Y, T41F, T41D, T41E, T41Q, T41N, and T41A. In a still morespecific aspect of this embodiment, the polypeptide has a T41Gsubstitution.

In another embodiment, the polypeptide of the invention is a variant ofSEQ ID NO:2 having a T41 substitution selected from T41G, T41W, T41H,T41V, T41I, T41L, T41R, T41K, T41Y, T41F, T41D, T41E, T41Q, T41N, andT41A; and only 2 additional amino acid substitutions selected from anyof the specific two amino acid substitutions set forth in Table 4. In aspecific aspect of this embodiment, the polypeptide has a T41Gsubstitution. In a more specific aspect of this embodiment, thepolypeptide has a T41G substitution. In another more specific aspect ofthis embodiment, one additional amino acid substitution is in epitope 1and the other additional amino acid substitution is in epitope 3. In astill more specific aspect of this embodiment, one additional amino acidsubstitution is T56H and the other additional amino acid substitution isK174R. In an even more specific aspect of this embodiment, thepolypeptide has the amino acid sequence set forth in SEQ ID NO:5

In another embodiment, the polypeptide of the invention is a variant ofSEQ ID NO:2 having a T41 substitution selected from T41G, T41W, T41H,T41V, T41I, T41L, T41R, T41K, T41Y, T41F, T41D, T41E, T41Q, T41N, andT41A; and only 3 additional amino acid substitutions selected from anyof the specific three amino acid substitutions set forth in Table 5. Ina specific aspect of this embodiment, the polypeptide has a T41Gsubstitution.

Nucleic Acid Molecules, Sequences, Vectors and Host Cells

In other embodiments, the invention provides an isolated nucleic acidmolecule that comprises a nucleic acid sequence coding for any of thepolypeptides or fusion proteins comprising a g3p variant describedabove. In one aspect of this embodiment, the isolated nucleic acidmolecule comprises a variant of nucleotides 64-714 of SEQ ID NO:3 ornucleotides 64-708 of SEQ ID NO:5, that is modified by a codonsubstitution, an in-frame codon insertion or an in-frame codon deletionthat destroys the putative glycosylation site encoded by nucleotides181-189 of SEQ ID NOS:3 or 4 (corresponding to the amino acids NAT atamino acids 39-41 of SEQ ID NOS:1 or 3). In a more specific aspect ofthese embodiments, the variant of nucleotides 64-714 of SEQ ID NOS:3 or4 is modified by a codon substitution that destroys the putativeglycosylation site encoded by nucleotides 181-189 of SEQ ID NOS:3 or 4.In an even more specific aspect of these embodiments, the variant ofnucleotides 64-714 of SEQ ID NOS:3 or 4 is modified by a codonsubstitution at nucleotides 187-189 (which encodes T41 of SEQ ID NOS:1and 2) that encodes an amino acid substitution selected from T41G, T41W,T41H, T41V, T41I, T41L, T41R, T41K, T41Y, T41F, T41D, T41E, T41Q, T41N,and T41A. In an even more specific aspect of these embodiments, thesubstituted codon substitution is selected from gga, tgg, cat, gtt, att,ctt, agg, aaa, tat, ttc, gac, gag, cag, aat, and gct. In an even morespecific aspect of these embodiments, the variant of nucleotides 64-714of SEQ ID NOS:3 or 4 is modified by a codon substitution at nucleotides187-189 that encodes the amino acid substitution T41G. In an even morespecific aspect of these embodiments, the substituted codon substitutionis gga.

In another embodiment, in addition to the modification that destroys theputative glycosylation site encoded by nucleotides 181-189 of SEQ IDNOS:3 or 4, the variant of nucleotides 64-714 of SEQ ID NOS:3 or 4further consists of 1-9 codon substitutions, wherein each codonsubstitution corresponds to an amino acid substitution selected from thesubstitutions set forth in Table 1, and Table 2, and any one of thefollowing V215 amino acid substitutions: V215A, V215S, V215G or V215T,V215C V215D, V215E, V215F, V215H, V215K, V215N, V215P, V215Q, and V215R.In an even more specific aspect of these embodiments the variant nucleicacid sequence is modified by one codon substitution selected to code forany one of the V215 amino acid substitutions set forth above; and from1-8 additional codon substitutions, wherein each of the additional codonsubstitutions is selected to code for an amino acid substitution setforth in Table 1. In a still more specific aspect of these embodimentsthe variant nucleic acid sequence is modified by one codon substitutionselected to code for any one of the V215 amino acid substitutions setforth above; and from 2-8 additional codon substitutions, wherein eachadditional codon substitutions encodes an amino acid substitution setforth in Table 1, and a codon substitution is present in each of atleast two of epitopes 1, 2 and 3. In a still more specific embodiment,the variant nucleic acid sequence is modified by one codon substitutionselected to code for any one of the V215 amino acid substitutions setforth above; and from 3-8 additional codon substitutions, wherein eachadditional codon substitution encodes an amino acid substitution setforth in Table 1, and a codon substitution is present in each ofepitopes 1, 2 and 3. In a still more specific embodiment, the variantnucleic acid sequence is modified by one codon substitution selected tocode for a V215A amino acid substitution; and one additional codonsubstitution selected to code for one of the single amino acidsubstitutions set forth in Table 3. In a more specific aspect of thisembodiment, the one additional codon substitution selected to code forone of the single amino acid substitutions set forth in Table 3 does notcode for an amino acid substitution in epitope 2. In another specificembodiment, the variant nucleic acid sequence is modified by one codonsubstitution selected to code for a V215A amino acid substitution setforth above; and two additional codon substitutions selected to code forone of the specific two amino acid substitutions set forth in Table 4.In a more specific aspect of this embodiment, the two additional codonsubstitutions selected to code for one of the specific two amino acidsubstitutions set forth in Table 4 does not code for an amino acidsubstitution in epitope 2. In a still more specific embodiment, thevariant nucleic acid sequence is modified by one codon substitutionselected to code for a V215 amino acid substitution set forth above; andthree additional codon substitutions selected to code for one of thespecific three amino acid substitutions set forth in Table 5.

In still other embodiments, the isolated nucleic acid molecule comprisesa variant of nucleotides 64-1530 of SEQ ID NO:3 or nucleotides 64-1524of SEQ ID NO:6, wherein the sequence is modified by a codonsubstitution, an in-frame codon insertion or an in-frame codon deletionthat destroys the putative glycosylation site encoded by nucleotides181-189 of SEQ ID NOS:3 or 4 (corresponding to the amino acids NAT atamino acids 39-41 of SEQ ID NOS:1 or 3). In a more specific aspect ofthese embodiments, the variant of nucleotides 64-1530 of SEQ ID NO:3 ornucleotides 64-1524 of SEQ ID NO:4 is modified by a codon substitutionthat destroys the putative glycosylation site encoded by nucleotides181-189 of SEQ ID NOS:3 or 4. In an even more specific aspect of theseembodiments, the variant of nucleotides 64-1530 of SEQ ID NO:3 ornucleotides 64-1524 of SEQ ID NO:4 is modified by a codon substitutionat nucleotides 187-189 (aca, which encodes T41 of SEQ ID NOS:1 and 2)that encodes an amino acid substitution selected from T41G, T41 W, T41H,T41V, T41I, T41L, T41R, T41K, T41Y, T41F, T41D, T41E, T41Q, T41N, andT41A. In an even more specific aspect of these embodiments, thesubstituted codon substitution is selected from gga, tgg, cat, gtt, att,ctt, agg, aaa, tat, ttc, gac, gag, cag, aat, and gct. In an even morespecific aspect of these embodiments, the variant of nucleotides 64-714of SEQ ID NOS:3 or 4 is modified by a codon substitution at nucleotides187-189 that encodes the amino acid substitution T41G. In an even morespecific aspect of these embodiments, the substituted codon substitutionis gga

In another embodiment, in addition to the modification that destroys theputative glycosylation site encoded by nucleotides 181-189 of SEQ IDNOS:3 or 4, the variant of nucleotides 64-1530 of SEQ ID NO:3 ornucleotides 64-1524 of SEQ ID NO:4 further consists of 1-9 codonsubstitutions, wherein each codon substitution corresponds to an aminoacid substitution selected from the substitutions set forth in Table 1,Table 2, and any one of the following V215 amino acid substitutions:V215S, V215G or V215T, V215C, V215D, V215E, V215F, V215H, V215K, V215N,V215P, V215Q, and V215R. Ina more specific aspect of this embodiment,each codon substitution corresponds to an amino acid substitutionselected from the substitutions set forth in Table 1, and any one of theV215 substitutions set forth above. In an even more specific embodiment,the variant nucleic acid sequence is modified by one codon substitutionselected to code for any one of the V215 amino acid substitutions netforth above and from 1-8 additional codon substitutions, wherein each ofthe additional codon substitutions corresponds to an amino acidsubstitution selected from the substitutions set forth in Table 1. In amore specific aspect, the variant has one additional codon substitutioncorresponding to one of the specific one amino acid substitutions setforth in Table 3. In a more specific aspect of this embodiment, the oneadditional codon substitution selected to code for one of the singleamino acid substitutions set forth in Table 3 does not code for an aminoacid substitution in epitope 2.

In another embodiment, in addition to the modification that destroys theputative glycosylation site encoded by nucleotides 181-189 of SEQ IDNOS:3 or 4, the variant of nucleotides 64-1530 of SEQ ID NO:3, ornucleotides 64-1524 of SEQ ID NO:6 has a modification that consists ofone codon substitution selected to code for any one of the V215 aminoacid substitution set forth above; and from 2-8 additional codonsubstitutions, wherein each additional codon substitution corresponds toan amino acid substitution set forth in Table 1, and a codonsubstitution is present in each of at least two of epitopes 1, 2 and 3.In a more specific aspect, the variant has two additional codonsubstitutions corresponding to one of the specific two amino acidsubstitutions set forth in Table 4. In a more specific aspect of thisembodiment, the two additional codon substitutions selected to code forone of the specific two amino acid substitutions set forth in Table 4does not code for an amino acid substitution in epitope 2. In an evenmore specific aspect of this embodiment, the specific two amino acidsubstitutions from Table 4 is T56H and K174R. In an even more specificaspect of this embodiment, the variant nucleotide sequence is SEQ IDNO:8.

In another embodiment, in addition to the modification that destroys theputative glycosylation site encoded by nucleotides 181-189 of SEQ IDNOS:3 or 4, the variant of any one of nucleotides 64-1530 of SEQ IDNO:3, or nucleotides 64-1524 of SEQ ID NO:6, has a modification thatconsists of one codon substitution selected to code for any one of theV215 amino acid substitution set forth above; and from 3-8 additionalcodon substitutions, wherein each additional codon substitutioncorresponds to an amino acid substitution set forth in Table 1, and acodon substitution is present in each of epitopes 1, 2 and 3. In a morespecific aspect, the variant has three additional codon substitutionscorresponding to one of the specific three amino acid substitutions setforth in Table 5.

In still other embodiments of the nucleic acid molecules of theinvention, the nucleic acid molecule further comprises nucleic acidsequences encoding a signal sequence fused in phase and directly to the5′ cd of the nucleic acid sequence encoding the variant g3p. In oneaspect of these embodiments, the nucleic acid sequence encoding thesignal sequence is nucleotides 1-63 of SEQ ID NO:3.

The nucleic acid molecules of the invention encompass nucleic acidsequences that are degenerative to, but encode the same amino acidsequence as encoded by any of the nucleic acid nucleic acid moleculesdescribed above.

For recombinant production, any of the nucleic acid molecules of theinvention may be inserted into an appropriate expression vector whichcontains the necessary elements for the transcription and translation ofthe inserted coding sequence, or in the case of an RNA viral vector, thenecessary elements for replication and translation. The encoding nucleicacid is inserted into the vector in proper reading frame. Accordingly,the invention provides vectors comprising nucleic acid molecule andsequences of the invention. Such vectors include, but are not limitedto, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc.The choice of appropriate vector in which to clone the nucleic acidmolecules and sequences of the invention may be made by those of skillin the art using well-known knowledge of the compatibility of the vectorwith the chosen host cell in which to carry out expression. This may bedone in any of mammalian cells, plant cells, insect cells, bacterialcells, yeast cells, etc. Appropriate vectors for each of these celltypes are well-known in the art and are generally commerciallyavailable.

In another embodiment, the invention provides a host cell harboring thevector containing a nucleic acid molecule or nucleic acid sequence ofthe invention. Methods of transfecting or transforming or otherwisegetting a vector of the invention into a host cell are known in the art.A cell harboring the vector, when cultured under appropriate conditions,will produce the polypeptides of the invention. Specific examples ofvectors and cells used for the recombinant production of thepolypeptides of the invention are set forth in the example sectionbelow.

Pharmaceutical Compositions

In some embodiments, the invention provides a pharmaceutical compositioncomprising any polypeptide or fusion protein comprising a variant g3p,optionally together with a pharmaceutically acceptable carrier, diluentor excipient. A “pharmaceutical composition” refers to a therapeuticallyeffective amount of a composition as described herein with aphysiologically suitable carrier and/or excipient. A pharmaceuticalcomposition does not cause significant irritation to an organism. Thephrases “physiologically suitable carrier” and “pharmaceuticallyacceptable carrier” which may be used interchangeably refer to a carrieror a diluent that does not cause significant irritation to an organismand does not abrogate the biological activity and properties of theadministered composition. The term “excipient” refers to an inertsubstance added to a pharmaceutical composition to further facilitateadministration of an active ingredient. Examples, without limitation,include, for example, saline, calcium carbonate, calcium phosphate,various sugars and types of starch, cellulose derivatives, gelatin,vegetable oils, polyethylene glycols, and surfactants, including, forexample, polysorbate 20.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in a conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intocompositions which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen and upon the nature ofthe composition delivered (e.g., size and solubility of thepolypeptide). In one aspect of these embodiments, the pharmaceuticalcomposition is formulated for injection or infusion into the bloodstreamof a patient. In another aspect of these embodiments, the pharmaceuticalcomposition is formulated for direct administration to the brain orcentral nervous system of the patient, for example, by directintramedullary, intrathecal, or intraventricular injection.

The compositions described herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Pharmaceutical compositions for parenteral administration includeaqueous solutions of the composition in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asoily or water based injection suspensions. Suitable lipophilic solventsor vehicles include fatty oils such as sesame oil, or synthetic fattyacids esters such as ethyl oleate, triglycerides or liposomes. Aqueousinjection suspensions may contain substances, which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol or dextran. Optionally, the suspension may also containsuitable stabilizers or agents (e.g., surfactants such as polysorbate(Tween 20)) which increase the solubility of the active ingredients toallow for the preparation of highly concentrated solutions. A proteinbased agent such as, for example, albumin may be used to preventadsorption of polypeptide of the invention to the delivery surface(i.e., IV bag, catheter, needle, etc.).

For oral administration, the compositions can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art.

Formulations may be presented in unit dosage form, e.g., in vials,ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents. Singledosage forms may be in a liquid or a solid form. Single dosage forms maybe administered directly to a patient without modification or may bediluted or reconstituted prior to administration. In certainembodiments, a single dosage form may be administered in holus form,e.g., single injection, single oral dose, including an oral dose thatcomprises multiple tablets, capsule, pills, etc. In alternateembodiments, a single dosage form may be administered over a period oftime, such as by infusion, or via an implanted pump, such as an ICVpump. In the latter embodiment, the single dosage form may be aninfusion bag or pump reservoir pre-filled with the appropriate amount ofa polypeptide or fusion protein comprising a variant g3p. Alternatively,the infusion bag or pump reservoir may be prepared just prior toadministration to a patient by mixing an appropriate dose of the variantg3p with the infusion bag or pump reservoir solution.

Another aspect of the invention includes methods for preparing apharmaceutical composition of the invention. Techniques for formulationof drugs may be found, for example, in “Remington's PharmaceuticalSciences,” Mack Publishing Co., Easton, Pa., latest edition, which isincorporated herein by reference in its entirety.

Pharmaceutical compositions suitable for use in the context of thepresent invention include compositions wherein the active ingredientsare contained in an amount effective to achieve the intended purpose.

Determination of a therapeutically or diagnostically effective amount iswell within the capability of those skilled in the art, especially inlight of the detailed disclosure provided herein.

Dosage amount and interval may be adjusted individually to provide brainlevels of the phage display vehicle which are sufficient to treat ordiagnose a particular brain disease, disorder, or condition (minimaleffective concentration, MEC). The MEC will vary for each preparation,but can be estimated from in vitro data. Dosages necessary to achievethe MEC will depend on individual characteristics.

Dosage intervals can also be determined using the MEC value.Preparations should be administered using a regimen, which maintainsbrain levels above the MEC for 10-90% of the time, preferable between30-90% and most preferably 50-90%.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated or diagnosed, the severity of theaffliction, the judgment of the prescribing physician, etc. In certainembodiments, the amount of polypeptide to be administered is selectedfrom 0.1-100 mg/kg subject body weight; 0.5-50 mg/kg; 1-30 mg/kg; 1-10mg/kg; 3-30 mg/kg; 1-3 mkg/kg; 3.10 mg/kg; and 10-30 mg/kg. In someembodiments, the peptide is administered to the subject once a week,once every two weeks, once every three weeks, once every four weeks, oronce a month.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising a preparation of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition, as if further detailed above.

It is to be understood that both the foregoing and following descriptionare exemplary and explanatory only and are not restrictive of theinvention, as claimed.

Therapeutic Uses

Another aspect of the invention relates to the use of any of thepolypeptides, nucleic acid molecules, or compositions of the invention,in the treatment of protein misfolding diseases, including, but notlimited to, those diseases involving any of: fAβ42, fαsyn or ftau.

In the context of treatments, the terms “patient”, “subject” and“recipient” are used interchangeably and include humans as well as othermammals. In some embodiments, a patient is a human who is positive for abiomarker associated with a protein misfolding disease. In oneembodiment, the patient exhibits ß-amyloid deposits as detected by PETimaging with florbetapir.

The term “treating” and its cognates are intended to mean reducing,slowing, or reversing the progression of a disease in a patientexhibiting one or more clinical symptoms of a disease. “Treating” isalso intended to mean reducing, slowing, or reversing the symptoms of adisease in a patient exhibiting one more clinical symptoms of a disease.In one embodiment, the patient exhibits ß-amyloid deposits as detectedby PET imaging with florbetapir and the number of ß-amyloid deposits isreduced by the treatment. In one embodiment, the patient exhibitsß-amyloid deposits as detected by the polypeptide or polypeptidecompositions of the present invention and the number of ß-amyloiddeposits are reduced or maintained by the treatment. In anotherembodiment, the patient exhibits any type of amyloid deposits asdetected by PET imaging and the cognitive function of the patient isimproved by the treatment. Improvement in cognitive function may beassayed by the methods and tests of McKhann et al., Alzheimer's &Dementia 7(3):263-9(2011).

“Prophylaxis” is distinct from treating and refers to administration ofa composition to an individual before the onset of any clinicalsymptoms. Prophylaxis using any of the polypeptides or compositionsthereof of the present invention is encompassed. Prophylaxis may beimplicated in individuals who are known to be at increased risk for adisease, or whom are certain to develop a disease, solely on the basisof one or more genetic markers. Many genetic markers have beenidentified for the various protein misfolding diseases. For examples,individuals with one or more of the Swedish mutation, the Indianamutation, or the London mutation in human amyloid precursor protein(hAPP) are at increased risk for developing early-onset Alzheimer'sDisease and so are candidates for prophylaxis. Likewise, individualswith the trinucleotide CAG repeats in the huntingtin gene, particularlythose with 36 or more repeats, will eventually develop Huntington'sDisease and so are candidates for prophylaxis.

The term “protein misfolding” refers to diseases characterized byformation of amyloid protein by an aggregating protein (amyloid formingpeptide), such as, but not limited to, ß-amyloid, serum amyloid A,cystatin C, IgG kappa light chain, or a prion protein. Diseases known tobe associated with misfolded and/or aggregated amyloid protein includeAlzheimer's disease, which includes early onset Alzheimer's disease,late onset Alzheimers disease, and presymptomatic Alzheimer's disease,Parkinson's disease, SAA amyloidosis, cystatin C, hereditary Icelandicsyndrome, senility, multiple myeloma, prion diseases including but notlimited to kuru. Creutzfeldt-Jakob disease (CJD),Gerstmann-Straussler-Scheinker disease (GSS), fatal familial insomnia(FFI), scrapie, and bovine spongiform encephalitis (BSE); amyotrophiclateral sclerosis (ALS), spinocerebellar ataxia (SCA1), (SCA3), (SCA6),(SCA7), Huntington disease, entatorubral-pallidoluysian atrophy, spinaland bulbar muscular atrophy, hereditary cerebral amyloid angiopathy,familial amyloidosis, frontotemporal lobe dementia. British/Danishdementia, Progressive Supranuclear Palsy (PSP), and familialencephalopathy. The polypeptides and compositions of the invention maybe used to treat “protein misfolding” diseases.

Many of these misfolded and/or aggregated amyloid protein diseases occurin the central nervous system (CNS). Some examples of diseases occurringin the CNS are Parkinson's Disease; Alzheimer's Disease; frontotemporaldementia (FTD) including those patients having the following clinicalsyndromes: behavioral variant FTD (bvFTD), progressive non-fluentaphasia (PNFA) and semantic dementia (SD); frontotemporal lobardegenerations (FTLDs); and Huntington's Disease. The polypeptides andcompositions of the invention may be used to treat diseasescharacterized by misfolded and/or aggregated amyloid protein that occurin the central nervous system (CNS).

Misfolding and/or aggregation of proteins may also occur outside theCNS. Amyloidosis A (AA) (for which the precursor protein is serum acutephase apolipoprotein, SAA) and multiple myeloma (precursor proteinsimmunoglobulin light and/or heavy chain) are two widely known proteinmisfolding and/or aggregated protein diseases that occur outside theCNS. Other examples include disease involving amyloid formed byα2-microglobulin, transthyretin (Familial Amyloidotic Polyneuropathy[FAP], Familial Amyloidotic Cardiomyopathy [FAC], and Senile SystemicAmyloidosis [SSA]), (apo)serum AA, apolipoproteins AT, AT, and ATV,gelsolin (Finnish form of Familial Amyloidotic Polyneuropathy),lysozyme, fibrinogen, cystatin C (Cerebral Amyloid Angiopathy,Hereditary Cerebral Hemorrhage with Amyloidosis, Icelandic Type),(pro)calcitonin, islet amyloid polypeptide (IAPP amyloidosis), atrialnatriuretic factor, prolactin, insulin, lactahedrin, kerato-epithelin,lactoferrin, odontogenic ameloblast-associated protein, and semenogelinI. The polypeptides and compositions of the invention may be used totreat diseases involving misfolding and/or aggregation of proteins thatoccur outside the CNS.

Neurodegenerative diseases may also involve tau lesions. Reviewed in Leeet al., Annu. Rev. Neurosci. 24:1121-159 (2001). Tau proteins aremicrotubule-associated proteins expressed in axons of both central andperipheral nervous system neurons. Neurodegenerative tauopathies(sometimes referred to as tauopathies) are encompassed. Examples oftauopathies include Alzheimer's Disease, Amyotrophic lateralsclerosis/parkinsonism-dementia complex. Argyrophilic grain dementia,Corticobasal degeneration, Creutzfeldt-Jakob disease, Dementiapugilistica, diffuse neurofibrillary tangles with calcification, Down'ssyndrome, Frontotemporal dementias including frontotemporal dementiawith parkinsonism linked to chromosome 17,Gerstmann-Sträussler-Scheinker disease, Hallervorden-Spatz disease,Myotonic dystrophy, Niemann-Pick disease type C, Non-Guamanian motorneuron disease with neurofibrillary tangles, Pick's disease,Postencephalitic parkinsonism, Prion protein cerebral amyloidangiopathy, Progressive subcortical gliosis, Progressive supranuclearpalsy, Subacute sclerosing panencephalitis, and Tangle only dementia.Some of these diseases may also include deposits of fibrillar amyloid βpeptides. For example. Alzheimer's disease exhibits both amyloid βdeposits and tau lesions. Similarly, prion-mediated diseases such asCreutzfeldt-Jakob disease, prion protein cerebral amyloid angiopathy,and Gerstmann-Sträussler-Scheinker syndrome may have also have taulesions. Thus an indication that a disease is a “tauopathy” should notbe interpreted as excluding the disease from other neurodegenerativedisease classifications or groupings, which are provided merely as aconvenience. The polypeptides and compositions of the invention may beused to treat neurodegenerative diseases as well as diseases involvingtau lesions.

In one embodiment, a pharmaceutical composition or formulation is foruse in a method of reducing amyloid in a patient exhibiting symptomsrelated to the presence of amyloid or that is positive for a biomarkerassociated with a protein misfolding disease, such as florbetapir(AV-45, Eli Lilly), comprising administering to the patient an effectiveamount of a pharmaceutical composition or formulation as describedherein. In one embodiment, the route of administration is selected fromintrathecal injection or infusion, direct intraventricular injection orinfusion, intraparenchymal injection or infusion, or intravenousinjection or infusion.

In one embodiment, a pharmaceutical composition or formulation is foruse in a method of maintaining the level of amyloid in a patientexhibiting symptoms related to the presence of amyloid or that ispositive for a biomarker associated with a protein misfolding disease,such as florbetapir (AV-45, Eli Lilly), comprising administering to thepatient an effective amount of a pharmaceutical composition orformulation as described herein. In one embodiment, the route ofadministration is selected from intrathecal injection or infusion,direct intraventricular injection or infusion, intraparenchymalinjection or infusion, or intravenous injection or infusion.

In one embodiment, a pharmaceutical composition or formulation is foruse in a method of disaggregating amyloid in a patient comprisingadministering to a patient having amyloid an effective amount of apharmaceutical composition or formulation as described herein. In oneembodiment, the route of administration is selected from intrathecalinjection or infusion, direct intraventricular injection or infusion,intraparenchymal injection or infusion, or intravenous injection orinfusion.

In one embodiment, a pharmaceutical composition or formulation of theinvention is for use in a method of causing the disaggregation ofß-amyloid deposits in the brain, comprising injecting directly into thebrain of a patient in need thereof an effective amount of pharmaceuticalcomposition as described herein, thereby causing a reduction inß-amyloid deposits in the brain. In an alternate embodiment, apharmaceutical composition or formulation of the invention is for use ina method of causing the disaggregation of ß-amyloid deposits in thebrain, comprising injecting intravenous delivery into a patient in needthereof an effective amount of pharmaceutical composition as describedherein, thereby causing a reduction in ß-amyloid deposits in the brain.

In one embodiment, a pharmaceutical composition or formulation is foruse in a method of reducing amyloid formation in the brain. Reducingamyloid formation in the brain may prevent, treat or reduce the symptomsor severity of a protein-misfolding or neurodegenerative disease. In oneembodiment, the route of administration is selected from intrathecalinjection or infusion, direct intraventricular injection or infusion,intraparenchymal injection or infusion, or intravenous injection orinfusion.

In one embodiment, a pharmaceutical composition or formulation of theinvention is for use in a method for promoting amyloid clearance in thebrain. Promoting amyloid clearance may prevent, treat or reduce thesymptoms or severity of a protein-misfolding or neurodegenerativedisease. In one embodiment, the route of administration is selected fromintrathecal injection or infusion, direct intraventricular injection orinfusion, intraparenchymal injection or infusion, or intravenousinjection or infusion.

In one embodiment, a pharmaceutical composition or formulation of theinvention is for use in a method for inhibiting amyloid aggregation inthe brain. Inhibiting amyloid aggregation in the brain may prevent,treat or reduce the symptoms or severity of a protein-misfolding orneurodegenerative disease. In one embodiment, the route ofadministration is selected from intrathecal injection or infusion,direct intraventricular injection or infusion, intraparenchymalinjection or infusion, or intravenous injection or infusion.

In one embodiment, a pharmaceutical composition or formulation of theinvention is for use in a method for clearing toxic amyloid oligomers inthe brain. Clearing toxic amyloid oligomers in the brain may prevent,treat or reduce the symptoms or severity of a protein-misfolding orneurodegenerative disease. In one embodiment, the route ofadministration is selected from intrathecal injection or infusion,direct intraventricular injection or infusion, intraparenchymalinjection or infusion, or intravenous injection or infusion.

In one embodiment, a pharmaceutical composition or formulation of theinvention is for use in a method for preventing the formation of toxicamyloid oligomers in the brain. Preventing the formation of toxicoligomers in the brain may prevent, treat or reduce the symptoms orseverity of a protein-misfolding or neurodegenerative disease. In oneembodiment, the route of administration is selected from intrathecalinjection or infusion, direct intraventricular injection or infusion,intraparenchymal injection or infusion, or intravenous injection orinfusion.

In one embodiment, a pharmaceutical composition or formulation of theinvention is for use in a method for protecting neurons from amyloiddamage. Protecting neurons from amyloid damage may prevent, treat orreduce the symptoms or severity of a protein-misfolding orneurodegenerative disease. In one embodiment, the route ofadministration is selected from intrathecal injection or infusion,direct intraventricular injection or infusion, intraparenchymalinjection or infusion, or intravenous injection or infusion. In oneembodiment, a pharmaceutical composition or formulation of the inventionfor use in protecting neurons from amyloid damage is givenprophylactically.

In some embodiments, the patient is positive for a biomarker associatedwith a protein misfolding and/or aggregation disease. In one embodiment,the biomarker is florbetapir (AV45, Eli Lilly).

In some embodiments, the patient is exhibiting symptoms of aneurodegenerative disease that is associated with the presence ofamyloid. In various embodiments, the amyloid is any of fAβ42, fαsyn orftau.

In certain embodiments, the neurodegenerative disease is Parkinson'sdisease, Alzheimer's disease, or Huntington's disease. In oneembodiment, the neurodegenerative disease is Alzheimer's disease. In oneembodiment, the neurodegenerative disease is Alzheimer's disease and thepatient exhibits ß-amyloid as detected by the imaging agent florbetapir(AV-45, Eli Lilly).

In some embodiments, the patient is exhibiting symptoms of aprion-mediated disease.

In certain embodiments, the prion-mediated disease is chosen fromCreutzfeldt-Jakob disease, kuru, fatal familial insomnia, orGerstmann-Sträussler-Scheinker syndrome.

In some embodiments, the patient is exhibiting symptoms of aneurodegenerative tauopathy other than Alzheimer's disease. In certainembodiments, the disease to be treated is selected from Argyrophilicgrain dementia, Corticobasal degeneration, Dementia pugilistica, diffuseneurofibrillary tangles with calcification, Down's syndrome,Frontotemporal dementias including frontotemporal dementia withparkinsonism linked to chromosome 17, Hallervorden-Spatz disease,Myotonic dystrophy, Niemann-Pick disease type C, Non-Guamanian motorneuron disease with neurofibrillary tangles, Pick's disease,Postencephalitic parkinsonism, Progressive subcortical gliosis,Progressive supranuclear palsy, Subacute sclerosing panencephalitis, andTangle only dementia.

In another embodiment, any of the disease conditions described above maybe treated by administration of a nucleic acid molecule of the invention(i.e., one that encodes a variant g3p that exhibits reducedimmunogenicity and possessing the ability to bind to amyloid,disaggregate amyloid plaques, and/or prevent aggregation of amyloid)alone or associated with a suitable carrier, such as, e.g., a lipidnanoparticle, a polymeric carrier, or a vector, such as a viral vectordirectly to a patient by any suitable route, such as, e.g., inhalationand intravenous infusion. The nucleic acid molecule encoding the variantg3p of the invention suitable for this treatment may be DNA or RNA.

Diagnostics

In another aspect of the invention, the polypeptides and compositionsdescribed herein, are used in diagnostic applications associated withthe various diseases described herein. For example, binding of acomposition of the invention when used as an imaging agent either invivo or in vitro may be part of a diagnosis of one of the proteinmisfolding diseases described. When used as diagnostic agents, thepolypeptides of the invention may further comprise a detectable label,or may be otherwise detected in vivo. Various labels can be attached tothe amyloid binding component of the diagnostic composition usingstandard techniques for labeling proteins. Examples of labels includefluorescent labels and radiolabels. There are a wide variety ofradiolabels that can be used, but in general the label is often selectedfrom radiolabels including, but not limited to, ¹⁸F, ¹¹C, and ¹²³I.These and other radioisotopes can be attached to the protein using wellknown chemistry. In one embodiment, the label is detected using positronemission tomography (PET). However, any other suitable technique fordetection of radioisotopes may also be used to detect the radiotracer.

The polypeptides and compositions of the invention may be used asdiagnostic imaging agents in combination with an imaging agent that isspecific for ß-amyloid such as, for example, F18-AV-45, Eli Lilly. Sincethere are currently no known imaging agents for non-ß-amyloidaggregates, the use of a diagnostic composition of the inventiontogether with a ß-amyloid-specific imaging agent will result in thedetection of non-ß-amyloid aggregates based on differential detection.Thus, in one embodiment, a diagnostic composition of the invention isused as an imaging agent in combination with a ß-amyloid imaging agentto detect non-ß-amyloid aggregates.

In another embodiment, the polypeptides or compositions of the inventionis used as a diagnostic imaging agent to detect ß-amyloid in the CNS,including the brain.

Diagnostic compositions of the invention may be administered using thesame routes described for therapeutic compositions. In one embodiment,the route of administration is selected from intrathecal injection orinfusion, direct intraventricular injection or infusion,intraparenchymal injection or infusion, or intravenous injection orinfusion.

EXAMPLES Example 1: Mapping of CD4+ T Cell Epitopes in g3p

87 overlapping peptides spanning the sequence of amino acids 1-240 ofSEQ ID NO:1 (15 amino acids long with 12 amino acid overlaps) weresynthesized and tested in a T cell epitope mapping assay for responsesfrom human CD4+ T cells. Individual peptides were tested in sextuplicatePBMC cultures and T cell responses were assessed in order to identifythe location of epitopes as well as their relative potency.

PBMC (peripheral blood mononuclear cells) were isolated from healthycommunity donor buffy coats (from blood drawn within 24 hours) obtainedfrom the UK National Blood Transfusion Service (Addenbrooke's Hospital,Cambridge, UK) and according to approval granted by Addenbrooke'sHospital Local Research Ethics Committee by Lymphoprep (Axis-shield,Dundee, UK) density centrifugation. CD8⁺ T cells were depleted usingCD8⁺ RosetteSep™ (StemCell Technologies Inc, London. UK). Donors werecharacterized by identifying HLA-DR haplotypes using an HLA SSP-PCRbased tissue-typing kit (Biotest, Solihull, UK). T cell responses to acontrol neoantigen protein (KLH protein (Pierce (Perbio), Cramlington,UK) and peptides derived from IFV and EBV) were also determined. PBMCwere then frozen and stored in liquid nitrogen until required.

A cohort of 55 donors was selected for the assay to best represent thenumber and frequency of HLA-DR allotypes expressed in the worldpopulation. Analysis of the allotypes expressed in the cohort againstthose expressed in the world population revealed that coverage of >80%was achieved and that all major HLA-DR alleles (individual allotypeswith a frequency >5% expressed in the world population) were wellrepresented. Details of individual donor haplotypes and a comparison ofthe frequency of MHC class II haplotypes expressed in the worldpopulation and the sample population are shown in Table 8 and FIG. 3,respectively.

TABLE 8 Donor details and haplotypes Donor No. Haplotype 1 DRB1*04:01,DRB1*16:01; DRB4*01:03; DQB1*03:02; DQB1*05:02 2 DRB1*01:01, DRB1*13:02;DRB3*03:01; DQB1*05:01; DQB1*06:04 3 DRB1*03:01, DRB1*07:01; DRB3*01:01;DRB4*01:03; DQB1*02:01; DQB1*03:03 4 DRB1*09:01, DRB1*13:01; DRB3*02:02;DRB4*01:03; DQB1*03:03; DQB1*06:03 5 DRB1*13:01, DRB1*13:02; DRB3*01:01;DRB3*03:01; DQB1*06:03; DQB1*06:04 6 DRB1*04:01, DRB1*04:07; DRB4*01:03;DQB1*03:01 7 DRB1*13:01; DRB3*01:01; DQB1*06:03 8 DRB1*13:01,DRB1*15:01; DRB3*02:02; DRB5*01:01; DQB1*06:02; DQB1*06:03 9 DRB1*04:01,DRB1*11:01; DRB3*02:02; DRB4*01:03; DQB1*03:01; DQB1*03:02 10DRB1*04:04, DRB1*12:01; DRB3*02:02; DRB4*01:03; DQB1*03:01; DQB1*03:0211 DRB1*13:02, DRB1*15:01; DRB3*01:01; DRB5*01:01; DQB1*06:02;DQB1*06:04 12 DRB1*04:01, DRB1*15:01; DRB4*01:03; DRB5*01:01;DQB1*03:02; DQB1*06:02 13 DRB1*04:02, DRB1*07:01; DRB4*01:01;DRB4*01:03; DQB1*02:01 14 DRB1*03:01, DRB1*16:01; DRB3*01:01;DRB5*02:02; DQB1*02:01; DQB1*05:02 15 DRB1*03:01, DRB1*13:01;DRB3*02:02; DQB1*02:01; DQB1*06:03 16 DRB1*01:01, DRB1*15:01;DRB5*01:01; DQB1*05:01; DQB1*06:02 17 DRB1*01:01, DRB1*07:01;DRB4*01:03; DQB1*03:03; DQB1*05:01 18 DRB1*01:01, DRB1*09:01;DRB4*01:03; DQB1*03:03; DQB1*05:01 19 DRB1*03:01, DRB1*11:02;DRB3*01:01; DRB3*02:02; DQB1*02:01; DQB1*03:01 20 DRB1*13:01;DRB3*01:01; DRB3*02:02; DQB1*06:03 21 DRB1*01:01, DRB1*13:02;DRB3*03:01; DQB1*05:01; DQB1*06:04 22 DRB1*04:01, DRB1*04:03;DRB4*01:03; DQB1*03:02 23 DRB1*08:01, DRB1*13:01; DRB3*01:01;DQB1*04:02; DQB1*06:03 24 DRB1*03:01, DRB1*15:01; DRB3*01:01;DRB5*01:01; DQB1*02:01; DQB1*06:02 25 DRB1*03:01, DRB4*01:01;DRB3*01:01; DRB4*01:03; DQB1*02:01; DQB1*03:01 26 DRB1*01:01,DRB1*15:01; DRB5*01:01; DQB1*05:01; DQB1*06:02 27 DRB1*04:04,DRB1*07:01; DRB4*01:01; DRB4*01:03; DQB1*02:02; DQB1*03:02. 28DRB1*11:01, DRB1*15:01; DRB3*02:01; DRB5*01:01; DQB1*03:01; DQB1*06:0129 DRB1*08:01, DRB1*15:01; DRB5*01:01; DQB1*04:02; DQB1*06:02 30DRB1*13:02, DRB1*15:01; DRB3*03:01; DRB5*01:01; DQB1*06:02; DQB1*06:0931 DRB1*04:01, DRB1*16:01; DRB4*01:03; DRB5*02:02; DQB1*03:02;DQB1*06:03 32 DRB1*13:02, DRB1*15:01; DRB3*03:01; DRB5*01:01;DQB1*06:02; DQB1*06:04 33 DRB1*07:01, DRB1*11:04; DRB3*02:02;DRB4*01:01; DQB1*02:02; DQB1*03:01 34 DRB1*01:03, DRB1*15:01;DRB5*01:01; DQB1*03:01; DQB1*06:02 35 DRB1*03:01, DRB1*14:01;DRB3*01:01; DRB3*02:02; DQB1*02:01; DQB1*05:03 36 DRB1*03:01,DRB1*08:01; DRB3*01:01; DQB1*02:01; DQB1*04:02 37 DRB1*03:01,DRB1*11:01; DRB3*01:01; DRB3*02:02; DQB1*02:01; DQB1*03:01 38DRB1*07:01, DRB1*15:01; DRB4*01:03; DRB5*01:01; DQB1*02;02; DQB1*06:0239 DRB1*03:01, DRB1*13:02; DRB3*02:02; DRB3*03:01; DQB1*02:01;DQB1*06:09 40 DRB1*01:01, DRB1*13:02; DRB3*01:01; DQB1*05:01; DQB1*06:0441 DRB1*04:07, DRB1*15:01; DRB4*01:03; DRB5*01:01; DQB1*03:01;DQB1*06:02 42 DRB1*07:01; DRB4*01:03; DQB1*02:02; DQB1*03:03 43DRB1*03:01, DRB1*15:01; DRB3*01:05; DRB5*01:01; DQB1*02:01; DQB1*06:0244 DRB1*07:01, DRB1*11:04; DRB3*02:02; DRB4*01:01; DQB1*02:02;DQB1*03:01 45 DRB1*03:01, DRB1*04:04; DRB3*01:01; DRB4*01:03;DQB1*02:01; DQB1*03:02 46 DRB1*04:04, DRB1*13:01; DRB3*02:02;DRB4*01:03; DQB1*03:02; DQB1*06:03 47 DRB1*04:01, DRB1*11:01;DRB3*02:02; DRB4*01:03; DQB1*03:01 48 DRB1*03:01, DRB1*04:01;DRB3*01:06; DRB4*01:03; DQB1*02:01; DQB1*03:02 49 DRB1*01:02,DRB1*13:03; DRB3*01:01; DQB1*03:01; DQB1*05:01 50 DRB1*04:07,DRB1*15:01; DRB4*01:03; DRB5*01:01; DQB1*03:01; DQB1*06:02 51DRB1*04:07, DRB1*13:02; DRB3*03:01; DRB4*01:03; DQB1*03:01; DQB1*06:0452 DRB1*03:01; DRB3*01:05; DQB1*02:01 53 DRB1*03:01, DRB1*07:01;DRB3*01:01; DRB4*01:01; DQB1*02:01; DQB1*02:02 54 DRB1*04:04,DRB1*15:01; DRB4*01:03; DQB1*03:02; DQB1*06:02 55 DRB1*03:01,DRB1*04:01; DRB3*01:01; DRB4*01:03; DQB1*02:01; DQB1*03:01

PBMC from each donor were thawed, counted and viability was assessed.Cells were revived in room temperature AIM-V® culture medium(Invitrogen. Paisley, UK) before adjusting the cell density to 2-3×10⁶PBMC/ml (proliferation cell stock). The 15 amino acid long peptides weresynthesized on a 1-3 mg scale with free N-terminal amine and C-terminalcarboxylic acid. Peptides were dissolved in DMSO to a concentration of10 mM and peptide culture stocks prepared by diluting into AIM-V®culture medium to a final concentration of 5 μM in the well. For eachpeptide and each donor, sextuplicate cultures were established in a flatbottomed 96 well plate. Both positive and negative control cultures werealso tested in sextuplicate. For each donor, three controls (KLH proteinand peptides derived from IFV and EBV) were also included. For apositive control, PHA (Sigma, Dorset, UK) was used at a finalconcentration of 2.5 μg/ml.

Cultures were incubated for a total of 6 days before adding 0.75 μCi³[H]-thymidine (Perkin Elmer®, Beaconsfield, UK) to each well. Cultureswere incubated for a further 18 hours before harvesting onto filter matsusing a TomTec Mach III cell harvester. Cpm for each well weredetermined by Meltilex™ (Perkin Elmer®, Beaconsfield, UK) scintillationcounting on a Microplate Beta Counter (Perkin Elmer®, Beaconsfield, UK)in paralux, low background counting mode.

For analysis of the data, a threshold of a stimulation index (SI) equalto or greater SI≥2.00 was used (with consideration of borderlineSI≥1.90-1.99 responses). Positive responses were defined by thefollowing statistical and empirical thresholds:

-   -   1. Significance (p<0.05) of the response by comparing cpm of        test wells against medium control wells using unpaired two        sample Student's t-test;    -   2. Stimulation index greater than 2.00 (SI≥2.00), where ST=mean        cpm of test wells/mean cpm medium control wells. Data presented        in this way is indicated as ST≥22.00, p<0.05.

In addition, intra-assay variation was assessed by calculating the CVand SD of the raw data from replicate cultures. Proliferation assayswere set up in sextuplicate cultures (“non-adjusted data”). To ensurethat intra-assay variability was low, the data were also analysed afterremoving the maximum and minimum cpm values (“adjusted data”) and the SIof donor responses was compared using both data sets. T cell epitopeswere identified by calculating the average frequency of positiveresponses (defined above) to all peptides in the study plus SD to give abackground response rate. Any peptide that induced proliferativeresponses above the background response rate in both the adjusted andnon-adjusted data was considered to contain a T cell epitope. When twooverlapping peptides induced a proliferative response rate the T-cellepitope was considered to be in the overlap region. Based upon this thefollowing T-cell epitopes were identified in the tested polypeptide:

Epitope 1: (amino acids 46-57 of SEQ ID NO: 1) C T G D E T Q C Y G T WEpitope 2:  (amino acids 133-144 of SEQ ID NO: 1)T F M F Q N N K F R N R Epitope 3:(amino acids of 172-183 of SEQ ID NO: 1) S S K A M Y D A Y W N GEpitope 4: (amino acids 214-225 of SEQ ID NO: 1) P V N A G G G S G G G SEpitope 5: (amino acids 253-267 of SEQ ID NO: 1)S G S G A M V R S D K T H T C

Example 2: Design of Substitutions in T Cell Epitopes 4 and 5 by InSilico Analysis

The sequences of peptides that were positive in the T cell assay wereanalysed using overlapping 9-mers from the epitope region using iTope™and TCED™ in silico technologies. [Perry et al., Drugs R D 9(6):385-96(2008).] Each 9-mer was tested against a database of MHC class IIalleles (34 in total) and scored based on the fit and interactions withthe MHC class II molecules. In addition, each 9-mer was BLAST searchedagainst a database of known CD4+ T cell epitopes in order to identifyany high sequence homology between that of the 9-mer and of databasepeptides from unrelated proteins that stimulated T cell responses inprevious T cell assays. On the basis of information from the in silicoanalysis, substitutions were identified for potential removal of CD4+ Tcell epitope activity from the identified epitopes.

Epitope 5 spans the C-terminus of the native N2-CT Gly-rich linker, theamino acids coded for by the multiple cloning site (“MCS”) of the pFUSEvector used to produce the N1-N2-human Ig Fc fusion protein of SEQ IDNO:1, and the N-terminus of the human Ig Fc region. In silico analysisimplicated M258 and V259 of SEQ ID NO:1 as the P1 anchors responsibleT-cell activity. Based on their location outside of the N1-N2 codingregion, removal of these two amino acids was not expected to cause aloss or function. These two amino acids were encoded by the MCS.Therefore, a double-stranded DNA molecule that modified the MCS andeliminated the nucleotides encoding M258 and V259 of SEQ ID NO:1 wasproduced by site-directed mutagenesis using appropriate oligonucleotideprimers. This was followed by recloning the resulting mutagenized DNAsequence back into the pFUSE vector using the using EcoRI and BglIIrestriction sites in the MCS. The resulting mature (lacking the signalsequence) fusion protein omitted M258 and V259. That fusion proteinretained the same ability to bind Abeta in the assay described below asthe SEQ ID NO:1 fusion protein.

Epitope 4 overlaps the N2 domain and the native Gly-rich linker. Crystalstructure of the g3p protein (not shown) suggested that Epitope 4 islocated away from amyloid binding region and therefore would be tolerantto amino acid substitutions without affecting activity. V215 (SEQ IDNO:1), which was identified as a P1 anchor, is surface exposed withslight orientation of side chain towards the protein core. Fromstructural analysis, any of the substitutions for V215 set forth inTables 6 and 7 should remove the epitope. In addition any of thesubstitutions of other amino acids within this epitope as set forth inTables 6 and 7 should also be accommodated. A nucleic acid sequenceencoding an N1-N2-Ig Fc comprising a V215A substitution (SEQ ID NO:4)and omitting M258 and V259 was derived from the above-describednucleotide sequence by site-directed mutagenesis using appropriateoligonucleotide primers. The resulting mature fusion protein (SEQ IDNO:2) demonstrated increased binding to Abeta in the binding assay ascompared to a fusion protein having the amino acid sequence of eitherSEQ ID NO:1 or the mature fusion protein lacking M258 and V239,described above. The nucleic acid sequence of SEQ ID NO:4 was used asthe parent sequence to crate genes incorporating all modifications inepitopes 1, 2 and 3.

Example 3: Design of Substitutions in T Cell Epitopes 1, 2 and 3 by InSilico Analysis

Epitope 1 lies just C-terminal to a putative Abeta binding portion ofN1-N2. In silica analysis of Epitope 1 highlighted amino acids 48-56 ofSEQ ID NO:1 as an area for amino acid substitution and removal of theT-cell epitope. Amino acids within this 9-mer were targeted forsubstitution based upon the nature of the existing amino acid, surfaceexposure, and interaction with the amyloid binding region of g3p, asinterpreted from the X-ray crystal structure of g3p. In particular, G48,T51, Y54 and T56 were targeted for substitution with the changesindicated in Table 1. Other potential amino acid substitutions in thisregion are set forth in Table 2.

iTope™ analysis of Epitope 2 pointed to amino acids 135-143 of SEQ IDNO:1 as a target for reducing or eliminating that epitope. Based on theX-ray crystal structure, amino acids 136-139 of SEQ ID NO:1 form a loopregion that forms bonds with the hinge region of N1-N2 and thus may beimportant for amyloid binding activity. Changes to these amino acids areless preferred and are only presented in Table 2. The more preferredchanges are to M135, R140, F141 and N143 and are set forth in Table 1.Other potential changes to this nine amino acid region are set forth inTable 2.

Amino acids 173-182 of SEQ ID NO:1 were identified within Epitope 3 astargets for substitution by in silico analysis. Epitope 3 is located inan alpha helical portion of the N2 domain, thus the strategy was toavoid introduction of hydrophobic residues and small polar unchargedresidues. In addition, we wanted to avoid introducing polar residuesacidic residues towards the C-terminus of this epitope. Based on X-raycrystallographic data, we targeted S173, D174, M176, D178 and W182 forsubstitution with the changes indicated in Table 1. Other potentialamino acid substitutions in this region are set forth in Table 2.

Example 4: Generation of N1-N2-Human IgG Fe Polypeptides Having ReducedT-Cell Epitopes

Fifty-eight different nucleic acid molecules, each encoding N1-N2-humanIgG Fc fusion proteins containing a different single amino acidsubstitution set forth in Table 3 were prepared. This was achieved bysite-directed mutagenesis of SEQ ID NO:4 using appropriateoligonucleotide primers to introduce the desired substitution, followedby recloning of the PCR-amplified mutagenized sequence into thepFUSE-hIgG1-Fc2 vector (Invivogen®, Toulouse, France, Catalogue No.pfuse-hg1fc2).

Genes encoding these “deimmunized” Fc fusion polypeptides weretransiently expressed in individual pFUSE-hIgG1-Fc2 vectors in FreeStyle293-F cells (Invitrogen, Paisley, Scotland. Catalogue #R790-07). On theday of transfection, cells were diluted to 1×10⁶/mL in FreeStyle 293Media (Invitrogen, Catalogue #12338) ensuring a viability of >90%.Plasmid DNA and polyethylenimine (PEI) were diluted separately inOptimem (Invitrogen, Catalogue #31985) and incubated for 5 minutesflowing which the PEI was added slowly to the DNA, and the DNA/PEImixtures were incubated for 5 minutes at room temperature. Afterincubation, the DNA/PEI mixtures were added dropwise to the 293-F cellswhilst swirling the flask. Transfected cultures were incubated at 37°C., 8% CO₂ on an orbital shaker platform rotating at 135 rpm for 6-7days, following which they were harvested.

Culture medium containing the polypeptide was harvested bycentrifugation and pH adjusted using 10×PBS. Proteins were bound toProtein A Sepharose beads (Sigma, Dorset, UK) by rotating overnight at4° C. The beads were washed twice with 1×PBS and transferred toSigmaPrep spin columns (Sigma). Samples were eluted by centrifugationusing 0.1M Glycine pH3.0 and neutralized in the collection tube using1/10^(th) volume 1M Tris-HCl pH8.0. Eluates were buffer exchanged into1×PBS using 2 ml ZebaSpin columns (Pierce, Cramlington, UK. Catalogue#89890). Samples were filter-sterilized and the absorbance at 280 nm wasmeasured for each sample.

Example 5: ABeta Binding Analysis of Deimmunized Polypeptides

A. ABeta (Aβ) Fiber Preparation.

Aβ42 (Img, rPeptide A-1002-2) was dissolved in hexafluoroisopropanol(HFIP, 1 mL), vortexed thoroughly and incubated at room temperature for2-18 hours until a clear solution appears. Aliquots (100 μl, 100 μg)were placed in 1.5 mL Eppendorf tubes and dry under vacuum (speed Vac,Eppendorf, Concentrator 5301) for 2-3 hr. The resulting monomers wereresuspended in 20 μL DMSO, pipetted and vortexed thoroughly untilcompletely dissolved. The solution was diluted with 260 μL of 10 mM HClsolution (final A42 concentration is 80 μM) and vortexed for 20 seconds.The clear solution is incubated (without shaking) for 3 days at 37° C.to allow for aggregation.

For use in the assay Aβ42 fibers from the resulting stock solution werediluted 50-fold to 1.6 μM final concentration in PBS.

B. ELISA Plate Preparation.

To each well of a 96-well plate (F96 MAXISORP NUNC-IMMUNO PLATE; Catalognumber: 442404, Lot 125436 and 128158; Denmark) was added 200 μL of a 1%BSA solution. The plates were sealed and incubated at 7° C. for 3 hr.Plates were then washed with PBS (250 μL/well)×3. We added 50 μL of thediluted Aβ42 fiber solution (1.6 μM) to each well and incubateduncovered at 37° C. overnight to complete dryness. PBS (50 μl/well) isadded to control wells (without Aβ42 fibers). Plates were then washed 2×with water and 1× with PBS (250 μL/well for each washing).

C. ELISA Assay.

Varying concentrations of each polypeptide (as well as the polypeptideof SEQ ID NO:2) in 50 μL were added to each well, as well as to non-Aβ42fiber coated wells and incubated for 1 h at 37° C. Plates were thenwashed 3× with PBS-T (0.05% Tween 20 in PBS) and 3× with PBS (250μL/well for each washing). We then added 50 μl of HRP-conjugated Goatanti-Human anti Fcγ (Jackson Labs, Catalog number, 109-035-008, Lotnumber: 106617) diluted 1:2500 (0.32 μg/mL final) in PBS-T+1% Milk(Difco™ Skim Milk, Becton, Dickinson and Company. USA, Catalog number:232100, Lot number: 7320448) to each well and incubated for 40 min at37° C. Plates were then washed 6× with PBS-T and 2× with PBS (250μL/well for each washing). We then added 50 μl/well OPD solution (5mg/7.5 ml 0.05 M Citrate buffer pH-5.513 μl H₂O₂) and let color todevelop for 3-6 min. We next added 25 μl/well of 4N HCl solution to stopreaction. Plates were read for absorbance at 492 nm and 405 nm. The 405nm absorbance was subtracted from the 492 nm absorbance and the resultsplotted as a function of polypeptide concentrations. An IC₅₀ for bindingfor each deimmunized polypeptide was then calculated and compared to theIC₅₀ calculated for the polypeptide of SEQ ID NO:2. The results areshown in Table 9, below.

TABLE 9 Relative Change in ABeta Binding IC₅₀ for Polypeptides with aSingle Additional Amino Acid Substitution in Epitope 1, 2 or 3 asCompared to Polypeptide of SEQ ID NO: 2 IC₅₀ Relative to Amino Acid SEQID Substitution NO: 2* Epitope 1 G48H 1.8 Epitope 1 G48K 1.1 Epitope 1G48R 1.9 Epitope 1 G48S 1.2 Epitope 1 G48T 1.0 Epitope 1 T51G 0.8Epitope 1 T51H 1.5 Epitope 1 T51K 2.5 Epitope 1 T51P 0.2 Epitope 1 T51R2.0 Epitope 1 T51Q 0.8 Epitope 1 T51N 0.5 Epitope 1 Y54G 0.02/0.2 Epitope 1 Y54H 0.3 Epitope 1 Y54K 0.13/0.32 Epitope 1 Y54P  0.07 Epitope1 Y54R 0.15/0.25 Epitope 1 T56G 0.1 Epitope 1 T56H 0.47/0.77 Epitope 1T56K  0.5/0.66 Epitope 1 T56P  0.1/0.09 Epitope 1 T56R 0.8 Epitope 2M135A 0.4 Epitope 2 M135D 0.5 Epitope 2 M135G 0.2 Epitope 2 M135H 0.1Epitope 2 M135K 0.4/0.2 Epitope 2 M135N 0.3 Epitope 2 M135R 0.1 Epitope2 M135T 0.14/0.3  Epitope 2 R140A 0.2 Epitope 2 R140D 0.3 Epitope 2R140E 0.3 Epitope 2 R140G 0.2 Epitope 2 R140H 0.2 Epitope 2 R140Q0.28/0.22 Epitope 2 F141D 0.2 Epitope 2 F141E 0.2 Epitope 2 N143A1.9/1.1 Epitope 2 N143G 0.19/0.08 Epitope 3 S173G 0.2 Epitope 3 S173P0.4 Epitope 3 M176G 0.3 Epitope 3 M176H 0.4 Epitope 3 M176K 0.2 Epitope3 M176N 0.5 Epitope 3 D178G 0.2 Epitope 3 D178N 0.5/0.4 Epitope 3 D178Q0.6 Epitope 3 D178S 0.3 Epitope 3 W181G 0.5 Epitope 3 W181H 0.47/0.87Epitope 3 W181K 0.3 Epitope 3 W181R 0.5/0.8 Epitope 3 S173K 0.17/0.07Epitope 3 K174R 1.2/1.0 Epitope 3 M176R 0.2 Epitope 3 D178T 0.4 *Numbersreflect IC₅₀ (substituted polypeptide)/IC₅₀ (polypeptide of SEQ ID NO:2). Multiple values reflect duplicate testing in the binding assay.

Example 6: Analysis of Whole Protein CD4+ T Cell Responses

In order to analyze CD4+ T cell responses from any of the polypeptidesof the invention in comparison to SEQ ID NO:1, a whole protein T cellassay was performed. PBMCs were isolated from 20 healthy human donorbuffy coats prepared as in Example 1. PBMCs were revived from frozen inAIM-V® culture medium and CD14⁺ cells were isolated using Miltenyi CD14Microbeads and LS columns (Miltenyi Biotech, Oxford, UK). Monocytes wereresuspended in ATM-V® supplemented with 1000 U/ml IL-4 and 1000 U/mlGM-CSF (“DC culture medium”) to 4-6×10⁶ PBMC/ml and then distributed in24 well plates (2 ml final culture volume). Cells were fed on day 2 byreplacement of a half volume DC culture medium. By day 3, monocytes haddifferentiated to semi-mature dendritic cells (DC) which werepre-incubated with antigens comprising either 40 ug/ml of testpolypeptide or 40 ug/ml of the polypeptide of SEQ ID NO:1 and 100 μg/mlKLH or medium only. Semi-mature DC were incubated with antigen for 24hours after which excess antigen was removed by washing the cells twiceand resuspending in DC culture medium supplemented with 50 ng/ml TNF-α(Peprotech, London, UK). DC were fed on day 7 by replacement of a halfvolume DC culture medium supplemented with 50 ng/mi TNFα and mature DCwere harvested on day 8. The harvested mature DC were counted andviability assessed using trypan blue dye exclusion. The DC were thenγ-irradiated (4000 rads) and resuspended at 2×10⁵ cells per ml in AIM-Vmedium before use analysis in T cell proliferation and ELISpot assays asbelow. Additionally, on day 8, fresh CD4+ T cells were also prepared. Topurify CD4+ T cells, PBMCs were revived in AIM-V® culture medium andCD4⁺ cells isolated using Miltenyi CD4 Microbeads and LS columns(Miltenyi Biotech, Oxford, UK) and resuspended in AIM-V® medium at 2×10⁶cells/ml.

On day 8, T cell proliferation assays were established whereby 1×10⁵autologous CD4⁺ T cells were added to 1×10⁴ antigen-loaded DC (ratio of10:1) in 96 well U-bottomed plates, with AIM-V® medium added to a finalvolume 200 ul/well. On day 14, assay plates were pulsed with 1 uCi [³H](Perkin Elmer, Beaconsfield, UK) per well in 25 ul AIM-V® for 6 hoursbefore harvesting onto filter mats (Perkin Elmer) using a TomTec MachIII (Hamden Conn., USA) cell harvester. All polypeptides were tested insextuplet cultures. Counts per minute (cpm) for each well weredetermined by Meltilex™ (Perkin Elmer) scintillation counting on a 1450Microbeta Wallac Trilux Liquid Scintillation Counter (Perkin Elmer) inparalux, low background counting. Counts per minute for each antigenwere normalised to the AIM-V® medium only control.

For ELiSpot assays, ELISpot plates (Millipore, Watford, UK) were coatedwith 100 ul/well IL-2 capture antibody (R&D Systems. Abingdon, UK) inPBS. Plates were then washed twice in PBS, incubated overnight in blockbuffer (1% BSA (Sigma) in PBS) and washed in AIM-V medium. On day 8,1×10⁵ autologous CD4⁺ T cells were added to 1×10⁴ antigen loaded DC(ratio of 10:1) in 96 well ELISpot plates. All polypeptide preparationswere tested in sextuplet cultures. For each donor PBMC, a negativecontrol (AIM-V® medium alone), no cells control and a PHA (10 ug/ml)positive control were also included.

After a further 7 day incubation period, ELISpot plates were developedby three sequential washes in dH₂O and PBS prior to the addition of 100ul filtered biotinylated detection antibody (R&D Systems, Abingdon, UK)in PBS/1% BSA. Following incubation at 37° C. for 1.5 hour, plates werefurther washed three times in PBS and 100 ul filtered streptavidin-AP(R&D Systems) in PBS/1% BSA was added for 1 hour (incubation at roomtemperature). Streptavidin-AP was discarded and plates were washed fourtimes in PBS. BCIP/NBT (R&D Systems) was added to each well andincubated for 30 minutes at room temperature. Spot development wasstopped by washing the wells and the backs of the wells three times withdH₂O. Dried plates were scanned on an Immunoscan™ Analyser and spots perwell (spw) were determined using Immunoscan™ Version 4 software.

For both proliferation and IL-2 ELISpot assays, results were expressedas a Stimulation Index (SI) defined as the ratio of cpm (proliferationassay) or spots (ELISpot assay) for the test polypeptide against amedium-only control using a threshold of SI equal to or greater than 2(SI≥2.0) for positive T cell responses.

Example 7: Design of Double and Triple Substitutions in Two or More of TCell Epitopes 1, 2 and 3

Based on the results of the binding assay, the following substitutionswere chosen at epitopes 1, 2 and 3 to be present in polypeptides thatcontain two amino acid substitutions as compared to SEQ ID NO:2, eachsubstitution in a different epitope.

TABLE 10 Amino Acid Substitutions for Variants Comprising Two Epitopeand Three Eptiope Modifications. Amino Original Amino Acid SubstitutionEpitope Acid in SEQ ID NO: 2 Amino Acids 1 54 Y K, R 1 56 T H, K 2 135 MK, T 2 140 R Q 3 174 K R 3 178 D N 3 181 W H, R

DNA encoding N1-N2-Human IG Fe fusion proteins having two of the aminoacid substitutions set forth in Table 10, each in a different epitope,were prepared by using site-directed mutagenesis of the appropriatestarting DNA (typically the DNA encoding for one of the twosubstitutions prepared as set forth in Example 3. The resulting DNAencoding these fusion proteins were used to transform cells and wereexpressed and purified as set forth in Example 4, and tested for bindingas set forth in Example 5. Polypeptides having one substitution in eachof epitopes 1, 2 and 3 were then designed based on the results of thebinding assay on the two amino acid substituted polypeptides.Polypeptides having one substitution in each of epitopes 1, 2 and 3 areassayed for both ABeta binding, as well as T-cell response as set forthin Example 6. In particular, the following double and triple epitopevariants were made by substituting certain amino acids in SEQ ID NO:2 asindicated in Table 11, below.

TABLE 11 Double and Triple Epitope Variant Polypeptides of theInvention. Polypeptide Starting Epitope 1 Epitope 2 Epitope 3 No.Sequence Substitution Substitution Substitution 63 SEQ ID NO: 2 Y54KM135K 64 SEQ ID NO: 2 Y54K M135T 65 SEQ ID NO: 2 Y54K R140Q 66 SEQ IDNO: 2 Y54R M135K 67 SEQ ID NO: 2 Y54R M135T 68 SEQ ID NO: 2 Y54R R140Q69 SEQ ID NO: 2 T56H M135K 70 SEQ ID NO: 2 T56H M135T 71 SEQ ID NO: 2T56H R140Q 72 SEQ ID NO: 2 T56K M135K 73 SEQ ID NO: 2 T56K M135T 74 SEQID NO: 2 T56K R140Q 75 SEQ ID NO: 2 Y54K D178N 76 SEQ ID NO: 2 Y54KW181H 77 SEQ ID NO: 2 Y54K W181R 78 SEQ ID NO: 2 Y54K K174R 79 SEQ IDNO: 2 Y54R D178N 80 SEQ ID NO: 2 Y54R W181H 81 SEQ ID NO: 2 Y54R W181R82 SEQ ID NO: 2 Y54R K174R 83 SEQ ID NO: 2 T56H D178N 84 SEQ ID NO: 2T56H W181H 85 SEQ ID NO: 2 T56H W181R 86 SEQ ID NO: 2 T56H K174R 87 SEQID NO: 2 T56K D178N 88 SEQ ID NO: 2 T56K W181H 89 SEQ ID NO: 2 T56KW181R 90 SEQ ID NO: 2 T56K K174R 91 SEQ ID NO: 2 M135K D178N 92 SEQ IDNO: 2 M135K W181H 93 SEQ ID NO: 2 M135K W181R 94 SEQ ID NO: 2 M135KK174R 95 SEQ ID NO: 2 M135T D178N 96 SEQ ID NO: 2 M135T W181H 97 SEQ IDNO: 2 M135T W181R 98 SEQ ID NO: 2 M135T K174R 99 SEQ ID NO: 2 R140QD178N 100 SEQ ID NO: 2 R140Q W181H 101 SEQ ID NO: 2 R140Q W181R 102 SEQID NO: 2 R140Q K174R

The above-indicated polypeptides were assayed for binding tobeta-amyloid using the ELISA assay set forth in Example 5. The resultsare set forth in Tables 12 and 13. Relative binding values reflect IC₅₀(polypeptide of SEQ ID NO:2)/IC₅₀ (tested polypeptide) (e.g., the lowerthe value the greater the binding of the polypeptide as compared to apolypeptide of SEQ ID NO:2). Multiple values reflect duplicate testingin the binding assay.

TABLE 12 Relative Binding Values of a Polypeptide of SEQ ID NO: 2 VersusExemplary Polypeptides of the Invention. Polypeptide Relative No.Binding Value 63 0.12 64 0.14 65 0.18 66 0.08 67 0.1  68 0.19 69 0.29,0.37 70 0.43, 0.45 71 0.42 72 0.40, 0.27 73 0.25, 0.39 74 0.26 75 0.1176 0.13 77 0.18, 0.16 78 0.24, 0.20 79 0.08 80 0.16 81 0.14 82 0.2  830.18, 0.36 84 0.26, 0.48 85 0.24, 0.79 86 0.51, 1.08 87 0.51, 0.83 880.65, 1.30 89 0.71, 1.05 90 1.43, 1.64 91 0.14 92 0.24 93 0.34 94 0.53,0.48 95 0.07 96 0.15 97 0.14 98 0.21, 0.61 99 0.11 100 0.36 101 0.2  1020.23

Example 8: Cellulose Acetate Filter Retardation Assay

This assay was used to monitor the destabilization (disaggregation) orremodeling of amyloid fibers into non-amyloidogenic or solubleaggregates. The assay was primarily adapted from Chang, E. and Kuret,J., Anal Biochem 373, 330-6, (2008) and Wanker, E. E. et al., MethodsEnzymol 309, 375-86, (1999). Specifically, 2.5 μM preparations of fAβamyloid fibers were pre-incubated with different concentrations of thevariant fusion polypeptides of the invention (1 nM to 2 μM) at 37° C.for 3 days. After incubation, fibers with and without fusion polypeptidewere diluted and spotted on cellulose acetate membranes on vacuum blots.The membranes were extensively washed with PBS and probed with anantibody specific for the N-terminal of Aß for 1 hr. HRP-conjugatedsecondary Ab was used to quantitate the fibrillar aggregates retained onthe membrane. Spot color was analyzed and digitized using adensitometric scanner. An EC₅₀ (half maximal effective concentration)was calculated based upon the intensities of the signal of each spotversus the concentration of fusion polypeptide added to each spot.

As can be seen from the above Examples, the variant polypeptides of theinvention all exhibited binding to Aß as determined by the ELISA assay.Most of the variant polypeptides tested also exhibited disaggregation ofAß, as determined by the dot blot assay.

Example 9: Construction and Analysis of Polypeptides with a ModifiedGlycosylation Signal

We constructed polypeptides lacking a glycosylation signal at aminoacids 39-41 of SEQ ID NO:1 or SEQ ID NO:2 using the nucleotides sequenceof cither SEQ ID NO:3 or a modified version of nucleotide sequence SEQID NO:4 that encoded Polypeptide No. 86 as starting material forsite-direct mutagenesis.

A plasmid vector derived from pFUSE-hIgG1-Fc2 vector (InVivogen) andencoding Polypeptide 86 fused to a mammalian signal sequence, wasmutagenized using the QuickChange Site-Directed Mutagenesis Kit(Agilent) and the following primers:

Forward primer: (SEQ ID NO: 8) GCTGTCTGTGGAATGCTGGAGGCGTTGTAGTTTGReverse primer: (SEQ ID NO: 9) CAAACTACAACGCCTCCAGCATTCCACAGACAGCfollowing manufacturer's directions to create a T41G substitution. Theresulting vector (SEQ ID NO:7) was used to transform NEB 5-alphacompetent E. coli cells in order to isolate and sequence the desiredplasmid using standard techniques.

The purified vector was then used to transform Expi293 cells using thecommercially available Expi293™ Expression System (Life Technologies).One day before transfection, Expi293 cells were seeded at a density of2×10⁶ viable cells/ml. On the day of transfection, 500 μg of thefilter-sterilized plasmid was diluted into Opti-MEM to a total volume of25 ml. In a separate tube, 1.333 ml ExpiFectamine™ 293 Reagent wasdiluted in 25 ml Opti-MEM I and mixed by inverting. After five minutesincubation at room temperature the diluted DNA was added to the dilutedExpiFectamine™ 293Reagent and incubated for an additional 20-30 minutes.The DNA-ExpiFectamine™ 293Reagent complex was slowly added to 500 mlcells (>3×10⁶ cells/ml) while gently swirling the flask. ExpiFectamine™293 Transfection Enhancers I and II, 2.5 ml and 25 ml respectively, wereadded to the transfected cells after approximately 18 hours and cellsare incubated for another 5 days at 37° C., 8% CO₂, 135 rpm on anorbital shaker. The expressed fusion protein (termed “Polypeptide86-T41G”)-containing media was harvested by centrifugation at 10,000 rpmat 4° C. for 20 minutes. The supernatant was purified on a 5 ml HiTraprProtein A FF column (GE Healthcare), with all steps being performed at4° C. The column was regenerated with 5 volumes of elution buffer (0.1Mglycine, pH 3), and washed in 5-10 volumes 20 mM sodium phosphate bufferbefore applying the cell media using a flow rate of 5 ml/min. The columnwas washed with 5-10 volumes 20 mM sodium phosphate buffer beforeeluting off bound Polypeptide 86-T41G with 0.1M glycine pH 3. One tothree ml fractions were collected in tubes with 1M Tris-HCl pH 9 toadjust pH. Yield was determined by absorbance at 280 nm on a Nanodrop2000C. Five μl of each protein-containing fraction was separated on aSDS-PAGE TGX gel (BioRad) and Coomassie stained for 2 hours. Fractionscontaining Polypeptide 86-T41G were pooled and dialyzed in D-PBSovernight at 4° C. The final Polypeptide 86-T41G sample was sterilizedon Ultrafree spin filters and the concentration was measured on theNanodrop 2000C.

Purified Polypeptide 86-T41G (SEQ ID NO:6) was analyzed by SDS-PAGE andmigrated as a single band with slightly lower molecular weight (apparent˜500 dalton less) than Polypeptide 86 (FIG. 9). We believe this lowermolecular weight is due to both the T to G change at amino acid 41, aswell as the loss of glycosylation on N39.

Purified Polypeptide 86-T41G was also analyzed by size exclusionchromatography on a Superdex200 increase 10/300 column. The column waswashed and equilibrated with 100 ml of phosphate buffered saline(“PBS”). One hundred micrograms (100 μg) of Polypeptide 86-T41G wasdiluted in PBS to a final volume of 200 μL and loaded onto the column.The column was then eluted with 1.5 column volumes of PBS at a rate of0.75 mL/minute. Protein in fractions was monitored byspectrophotometrically at 214 nm and 280 nm and demonstrated a sharppeak indicating homogeneity (data not shown).

Purified Polypeptide 86-T41G was analyzed for Abeta binding using theELISA described in Example 5. The EC₅₀ for Abeta binding in this assaywas calculated to be 13.15 nM, compared to 20.6-27.01 nM for thepolypeptide of SEQ ID NO:1 and 34.5 nM for Polypeptide 86.

Purified Polypeptide 86-T41G was also compared to the polypeptide of SEQID NO:1 and Polypeptide 86 for Abeta binding using the cellulose acetatefilter retardation assay described in Example 8. The results of thisassay are shown in FIG. 10.

Purified Polypeptide 86-T41G was then compared to Polypeptide 86 and thepolypeptide of SEQ ID NO:1 (as well as humanized A33 antibody andkeyhole limpet hemocyanin as positive controls) in the whole proteinCD4+ T cell Response assay using 50 different PBMC donors representing95% of the human HLA haplotypes; and in the ELISpot cytokine (IL-2)assays described in Example 6. The results are shown in Tables 13 and14, below.

TABLE 13 PBMC T-cell Proliferative Response Assay Results. Mean % SampleSI SD Response SEQ ID NO: 1 2.21 0.32 12 Polypeptide 86 2.66 1.02 4Polypeptide 86 T41G 2.11 0.15 4 Humanized A33 3.29 1.83 12 KLH 4.74 3.2884

TABLE 14 ELISpot IL-2 Assay Results. Mean % Sample SI SD Response SEQ IDNO: 1 2.51 0.64 14 Polypeptide 86 2.83 1.03 4 Polypeptide 86 T41G 2.330.22 4 Humanized A33 2.46 0.33 20 KLH 4.57 4.32 86

As can be seen from the above Tables the polypeptide of SEQ ID NO:1 (noamino acid changes in either the putative glycosylation site at aminoacids 39-41 or any putative T-cell epitopes) elicited proliferativeresponses (“SI”)>2 times background for 12% of the donors (6/50).Polypeptide 86 and Polypeptide 86T41G elicited proliferative responsesfrom significantly fewer donor PBMCs (4%; 2/50) with responders havingproliferative response also slightly higher than 2 times background.This indicates lower projected immunogenicity of Polypeptide 86 T41G forhuman subjects. The IL-2 assay confirms the T-cell response assayresults.

Polypeptide 86 T41G was also compared to the polypeptide of SEQ ID NO:1for binding to Abeta42 fibers. NAC fibers and tau-mtbr fibers.

Fiber and ELISA Plate Preparation.

Aβ42 peptide (rPeptide A-1002-2) was dissolved in hexafluoroisopropanolby vortexing and incubation at room temperature for 18 hours. Aliquotswere dried under vacuum and stored at −20° C. 100 ug of Aβ42 monomerswere dissolved in 20 μl DMSO, dissolved by vortex and diluted to 80 μMin 10 mM HCl solution. The Aβ42 peptide solution was incubated for 3days at 37° C. and fiber formation verified with ThT fluorescence assay.

The non-amyloid beta component (NAC) of senile plaque is an aggregatedfragment of alpha-synuclein, the aggregate that is the hallmark ofParkinson's disease. NAC peptide ((Bachem H2598) was dissolved in 20 mMNaHCO₃ at 600 uM and centrifuged for 1 hour, 100,000×g at 4° C.Supernatant was neutralized with 2N HCl and mixed 1:1 with 10 mM HCl.The peptide was incubated for 4 days at 37° C. and fiber formationconfirmed by ThT fluorescence assay.

Fibers comprising the microtubule binding portion of Tau (Tau-mtbrfibers) were made according to Frost et al. J Biol Chem. 2009 May 8;284(19):12845-52. Briefly, 40 uM of tau-mtbr protein was incubated with40 uM low-molecular weight heparin (Fisher Scientific, BP2524) and 2 mMDTT for 3 days at 37° C. Fibril formation was confirmed by ThTfluorescence assay.

Fibers were diluted to 1 μM in PBSA-0.02% and dry-coated on MaxisorpNunc Immunoplate ELISA plates (ThermoFisher Cat no. 442404) byincubation over night at 37° C. Wells were blocked, 200 μl/well, inSuperblock (ThermoFisher Cat no. 37515) for 1 hour at room temperatureand washed in PBST-0.05%.

Binding Assay and Results.

The polypeptide of SEQ ID NO:1 and Polypeptide 86 T41G were separatelyadded to the fiber ELISA at 50 and 200 nM and incubated for 1 hour at37° C. Wells were washed in PBST-0.05% 6×200 μl before incubation withgoat anti-human IgG Fc fragment specific-HRP (Jackson labs Cat no.109-035-008), 1:2500 in TBST-0.05%; 1% milk block (LabScientific Cat no.732-291-1940), for 45 minutes in room temperature. Plates were washed in4×200 ul TBST-0.05%, 2×200 ul PBS before adding 50 ul TMB solution(Sigma T0440) per well. The reaction was left to develop for 8 minutesand stopped by adding 50 μl 2N HCl per well. The absorbance at 450 nmwas recorded in a Tecan plate reader (Infinite M1000Pro).

Data points were taken from the average of triplicate wells withstandard deviation calculated with GraphPad Prism. The values werecorrected for background by subtracting the mean absorbance in wellsincubated without either polypeptide for each substrate.

As shown in FIG. 11, Polypeptide 86 T41G bind Aß42m NAC and tau-mtbrfibers with the same or higher affinity compared to the polypeptide ofSEQ ID NO:1.

We also constructed by similar protocols the following variants of SEQID NO:1 modified only to eliminate the putative glycosylation site(substitution indicated in parentheses):

Polypeptide 200 (N39A) Polypeptide 201 (N39Q) Polypeptide 202 (T41M)Polypeptide 203 (T41W) Polypeptide 204 (T41H) Polypeptide 205 (T41V)Polypeptide 206 (T41I) Polypeptide 207 (T41L) Polypeptide 208 (T41R)Polypeptide 209 (T41K) Polypeptide 210 (T41Y) Polypeptide 211 (T41F)Polypeptide 212 (T41D) Polypeptide 213 (T41E) Polypeptide 214 (T41Q)Polypeptide 215 (T41N) Polypeptide 216 (T41A) Polypeptide 217 (T41G).

Example 10: Pharmacokinetic (PK) Studies of Polypeptide 217 and SEQ IDNO:1

Animal Treatment and Sample Collection.

C57Bl6 mice (8-12 wks; Hilltop Lab Animals) were administered a single20 mg/kg intraperitoneal dose of the polypeptide of SEQ ID NO:1 (n=22)or Polypeptide 217 (n=22) used. The polypeptide of SEQ ID NO:1 wasadministered once (20 mg/kg, i.p.) to 22 mice. Polypeptide 217 wasadministered once (20 mg/kg, i.p.) to a separate set of 22 mice. Bloodwas collected once each animal at different times (0 h, 6 h, 9 h, 12 h,1 d, 3 d, 7 d and 14 d post-dosing). Plasma was isolated from the bloodsamples, stored in 100 μL aliquots, and used for all subsequentanalyses. After collection of the blood, mice were euthanized,transcardially perfused with PBS and their brains harvested. The brainswere hemisected and each hemisphere further sectioned into a rostral,caudal, hippocampus and cerebellum portion. Plasma was shipped toIntertek (San Diego, Calif.) for pharmacokinetic analysis, while leftfrontal cortex was shipped to Cambridge Biomedical (Boston, Mass.) forPK analysis.

Plasma ELISA Analysis.

All standards and samples that were analyzed were exposed to 217 mMacetic acid for 30 min at room temperature (“RT”), and then neutralizedin (1:1.5 v/v 1M Tris pH 9.5:sample). The acid dissociation stepsolubilized polypeptide present in an insoluble fraction.

A sandwich ELISA assay was used to measure polypeptide levels in plasma.MaxiSorp™ plates were coated with rabbit anti-M13 (Abcam: ab6188) at1:1,000 dilution from stock (3.7 μg/mL, 0.37 μg/well) overnight incarbonate buffer (pH 9.6) at 4° C. Plates were washed three times withPBS containing 0.1% Tween-20 (“PBST”) and blocked with 1% milk in PBSfor 2 h at 37° C. followed by 1 h at RT. Plates were again washed threetimes with PBST and then samples or standards were added to wells andincubated for 1 h at 37° C. Wells were then washed 3× with PBST, andincubated with HRP-labeled goat anti-Human IgG (heavy & light chains,Bethel: A80-219P; 1:10,000) for 30 min at RT. Wells were washed 3× withPBST, and the plates were then developed at RT with TMB substrate.Reactions were stopped after the A450 of the highest standards wasbetween 0.6-0.8. Levels of polypeptide were quantified from theabsorbance read at 450 nm, minus the reference absorbance at 650 nm.Plasma was analyzed at dilutions of 1:20, 1:300 and 1:3,000; no matrixinterference was observed at these dilutions. The results are shownbelow in Table 15.

TABLE 15 Plasma Pharmacokinetic Parameters. Parameter SEQ ID NO: 1Polypeptide 217 C_(max) 140 μg/mL 179 μg/mL T_(max) 6 h 6 h Beta-phase ½life 5 days 10 days Clearance 24.5 mL/day/kg 8.3 mL/day/kg AUC 816.33day*μg/ml 2396.1 day*μg/ml

Brain ELISA Analysis.

Brain tissue (left frontal cortex) was homogenized in cold PBS usingtrip Pure M-Bio Grade beaded tubes and a Precellys024 Lysis Homogenizer(5,000 RPM twice for 20 sec, with a 5 se interval between homogenizationcycles). Homogenate was centrifuged at 14,000 rpm for 5 min at 4° C.Supernatant was removed to a new tube and used for all subsequentanalyses. Protein content of brain lysate was determined using a PierceBCA protein assay kit. Lysate was used at a 1:2 dilution.

A sandwich ELISA assay was used to measure polypeptide levels in brain.MaxiSorp™ plates were coated with rabbit anti-M13 (Abcam: ab6188) at1:1,000 (3.7 μg/mL, 0.37 μg/well) overnight in carbonate buffer at 4° C.Plates were washed 3× with PBST and blocked with 1% milk in PBS for 2 hat 37° C., followed by 1 h at RT. Plates were then washed 3× with PBST,and samples or standards were added to wells and incubated for 1 h at37° C. Plates were again washed 3× with PBST, and then wells wereincubated with HRP-labeled donkey anti-Human IgG (heavy & light chains,Jackson ImmunoResearch: 709-035-149; 1:10,000) for 30 min at RT. After3× washes with PBST, plates were developed for 15 min at RT with TMBsubstrate. Reactions were stopped and absorbance read at 450 nm. Levelsof polypeptide in brain were expressed relative to protein content oflysates. The results are shown below in Table 16.

TABLE 16 Brain Pharmacokinetic Parameters Parameter SEQ ID NO: 1Polypeptide 217 C_(max) 2.5 ng/mg 2.7 ng/mg T_(max) 3 d 3 d Beta-phase ½life 3 days 7 days AUC 14.44 day*ng/mg 32.90 day*ng/mg

1-37. (canceled)
 38. A polypeptide comprising a variant of a startingamino acid sequence, wherein the polypeptide binds to and/ordisaggregates amyloid; wherein the starting amino acid sequence isselected from amino acids 1-217 of SEQ ID NO:1 or amino acids 1-217 ofSEQ ID NO:2: wherein the variant differs from the starting amino acidsequence by removal of the putative glycosylation signal at amino acids39-41; and wherein the variant optionally and additionally differs fromthe starting amino acids sequence by one or more of: (a) one or moremodifications selected from: (i) substitution of VVV at amino acids43-45 with AAA; (ii) substitution C53W; (iii) deletion of amino acids96-103; (iv) substitution of QPP at amino acids 212-214 with AGA; (v)substitutions W181A, F190A and F194A; (vi) deletion of amino acid 1;(vii) deletion of amino acids 1 and 2; and (viii) addition of aN-terminal methionine residue; and (b) decreased immunogenicityresulting from 1 to 9 amino acid substitutions selected from: Amino Acidpresent in the Amino Starting Amino Acid # Acid Sequence Amino AcidSubstitutions 48 G H, K, R, S, T, D, P 50 E G, H, K, P, R 51 T G, H, K,R, P, Q, N, W 53 C F, H, K, N, Q, R, W, Y 54 Y G, H, K, R, P 56 T G, H,K, R, P 135 M A, D, G, K, N, T, H, R, C, E, P, Q, S 137 Q D, E 138 N D,E, G, H, P, Q, S, T 140 R D, E, H, Q, A, G, M, N, P, S, Y 141 F D, E, G,N, P, Q, Y 143 N A, G 173 S G, P, K, D, H, R, T 174 K R 175 A G, H, K,P, R 176 M G, H, K, N, R, P, Q, W 178 D G, N, Q, S, T, F, H, K, R, W, Y179 A H, K, P, R 181 W G, H, K, R, P

wherein when the starting amino acid sequence is amino acids 1-217 ofSEQ ID NO:1, any of the 1 to 9 amino acid substitutions in (b) mayadditionally be selected from a substitution of V215 with S, T, C, D, E,F, H, K, N, P, Q, or R.
 39. The polypeptide according to claim 38,wherein the modification to remove the putative glycosylation signal atamino acids 39-41 of SEQ ID NO:1 or SEQ ID NO:2 is an amino acidsubstitution of N39.
 40. The polypeptide of claim 39, wherein themodification to remove the putative glycosylation signal at amino acids39-41 of SEQ ID NO:1 or SEQ ID NO:2 is an amino acid substitutionselected from T41G, T41W, T41H, T41V, T41I, T41L, T41R, T41K, T41Y,T41F, T41D, T41E, T41Q, T41N, and T41A.
 41. The polypeptide of claim 38,wherein each of the 1 to 9 amino acid substitutions of claim 38(b) isselected from: Amino Acid present in the Amino Starting Amino Acid #Acid Sequence Amino Acid Substitutions 48 G H, K, R, S, T 51 T G, H, K,R, P, Q, N 54 Y G, H, K, R, P 56 T G, H, K, R, P 135 M A, D, G, K, N, T,H, R 140 R D, E, H, Q, A, G 141 F D, E 143 N A, G 173 S G, P, K 174 K R176 M G, H, K, N, R 178 D G, N, Q, S, T 181 W G, H, K, R


42. The polypeptide of claim 38, wherein the starting amino acidsequence is modified by 2 to 9 amino acid substitutions selected fromclaim 38(b), wherein at least one substitution is present in epitope 1,comprising amino acids 48-56 of SEQ ID NO:1 or SEQ ID NO:2; and whereinat least one substitution is present in epitope 3, comprising aminoacids 173-181 of SEQ ID NO:1 or SEQ ID NO:2.
 43. The polypeptide ofclaim 38, wherein the starting amino acid sequence is modified by twoamino acid substitutions selected from claim 38(b) selected from thegroup of two amino acid substitutions set forth below: Y54K and D178NY54K and W181H Y54K and W181R Y54K and K174R Y54R and D178N Y54R andW181H Y54R and W181R Y54R and K174R T56H and D178N T56H and W181H T56Hand W181R T56H and K174R T56K and D178N T56K and W181H T56K and W181RT56K and K174R


44. The polypeptide of claim 38, consisting essentially of a human orhumanized immunoglobulin Fc polypeptide sequence fused either via apeptide linker or directly to the C-terminus of the variant of thestarting amino acid sequence.
 45. The polypeptide of claim 44, whereinthe immunoglobulin Fc polypeptide sequence is the Fc portion of a humanIgG.
 46. The polypeptide of claim 45, wherein the amino acid sequence ofthe peptide linker and Fc portion of human IgG is selected from aminoacids 218-488 of SEQ ID NO:1, and amino acids 218-486 of SEQ ID NO:2.47. A pharmaceutical composition comprising the polypeptide of claim 38and a pharmaceutically acceptable carrier.
 48. The pharmaceuticalcomposition of claim 47, wherein the composition is formulated forinjection into the bloodstream of a patient, infusion into thebloodstream of a patient, direct administration to the brain, or directadministration to the CNS.
 49. A method of reducing amyloid or tauprotein aggregates in a patient in need thereof, comprisingadministering to the patient an effective amount of the polypeptide ofclaim
 38. 50. The method of claim 49, wherein the patient is positivefor florbetapir when florbetapir is used as an imaging agent in positronemission tomography.
 51. The method of claim 49, wherein the patient issuffering from and/or exhibits symptoms of a disease selected fromAlzheimer's disease, early onset Alzheimer's disease, late onsetAlzheimer's disease, presymptomatic Alzheimer's disease, Parkinson'sdisease, SAA amyloidosis, disease characterized by formation of amyloidprotein by aggregation of cystatin C, disease characterized by formationof amyloid protein by aggregation of immunoglobulin light chain,familial amyloidotic polyneuropathy (FAP), familial amyloidoticcardiomyopathy (FAC), islet amyloid polypeptide (IAPP) amyloidosis,Finnish form of FAP (aggregation of gelsolin), senile systemicamyloidosis (SSA), hereditary Icelandic syndrome, senility, multiplemyeloma, prion diseases, kuru, Creutzfeldt-Jakob disease (CJD),Gerstmann-Straussler-Scheinker disease (GSS), fatal familial insomnia(FFI), scrapie, bovine spongiform encephalitis (BSE), amyotrophiclateral sclerosis (ALS), spinocerebellar ataxia (SCA1, SCA3, SCA6, orSCA7), Huntington's disease, dentatorubral-pallidoluysian atrophy,spinal and bulbar muscular atrophy, hereditary cerebral amyloidangiopathy, familial amyloidosis, British/Danish dementia, familialencephalopathy, Amyotrophic lateral sclerosis/parkinsonism-dementiacomplex, Argyrophilic grain dementia, Corticobasal degeneration,Dementia pugilistica, diffuse neurofibrillary tangles withcalcification, Down's syndrome, Hallervorden-Spatz disease, Myotonicdystrophy, Niemann-Pick disease type C, Non-Guamanian motor neurondisease with neurofibrillary tangles, Pick's disease, Postencephaliticparkinsonism, Prion protein cerebral amyloid angiopathy, Progressivesubcortical gliosis, Progressive supranuclear palsy, Subacute sclerosingpanencephalitis, Tangle only dementia, frontotemporal lobardegenerations (FTLDs), and frontotemporal lobe dementia (FTD) includinga patient having one or more of the following clinical syndromes:behavioral variant FTD (bvFTD), progressive non-fluent aphasia (PNFA),frontotemporal dementia with parkinsonism linked to chromosome 17,Progressive Supranuclear Palsy (PSP), and semantic dementia (SD).
 52. Anucleic acid sequence encoding the polypeptide of claim 38, wherein thenucleic acid sequence.
 53. The nucleic acid sequence of claim 52,wherein the nucleic acid sequence further encodes a mammalian signalsequence fused to and in frame with the polypeptide encoding sequence.54. A vector comprising a nucleic acid sequence of claim 52, wherein thenucleic acid sequence is operatively linked to an expression controlsequence in the vector.
 55. A host cell comprising the vector of claim54.
 56. A method of making a polypeptide of claim 38, comprising thesteps of expressing the protein encoded by the nucleic acid sequence ofclaim 52; and isolating the expressed polypeptide.
 57. A method ofmaking a polypeptide of claim 38, comprising the steps of culturing thehost cell of claim 55 under conditions sufficient to allow expression ofthe polypeptide; and isolating the expressed polypeptide.